Difference: ExpressMediaM9 (1 vs. 28)

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O
• 1 ml 1 M CaCl₂.5H₂O
• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

Metal Solutions

-- DavidCowburn - 14 Apr 2008

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O
• 1 ml 1 M CaCl₂.5H₂O
• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

Metal Solutions

-- DavidCowburn - 14 Apr 2008

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O
• 1 ml 1 M CaCl₂.5H₂O
• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

Metal Solutions

-- DavidCowburn - 14 Apr 2008

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O
• 1 ml 1 M CaCl₂.5H₂O
• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

Metal Solutions

Metal Sol-I

-- DavidCowburn - 14 Apr 2008

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O
• 1 ml 1 M CaCl₂.5H₂O
• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

Metal Solutions

Metal Sol-I

Metal Sol-II

-- DavidCowburn - 14 Apr 2008

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O

• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

Metal Solutions

Metal Sol-I

Metal Sol-II

-- DavidCowburn - 14 Apr 2008

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O
• 1 ml 1 M MgCl₂.5H₂O
• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

Metal Solutions

Metal Sol-I

Metal Sol-II

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O
• 1 ml 1 M MgCl₂.5H₂O
• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

Metal Solutions

Metal Sol-I

Metal Sol-II

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O

• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

M9 medium for unlabeled protein (15N or 15N, 13C) OR (14N, 12C)

Metal Solutions

Metal Sol-I

Metal Sol-II

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl

• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  

• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

M9 medium for unlabeled protein (15N or 15N, 13C) OR (14N, 12C)

Metal Solutions

Metal Sol-I

Metal Sol-II

• Ampicillin or carbenicillin

• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.

1. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.

1. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.

1. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.

1. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
   

M9 medium for unlabeled protein (15N or 15N, 13C) OR (14N, 12C)

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Casamino acids
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl, MgCl₂, thiamine, glucose or glycerol
• Escherichia coli BL21 (DE3) (Novagen) or derivatives
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 g NaCl

M9 medium For 1 l, combine the following:

• 6 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 g NaCl

• 0.7 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 M MgSO₄
• 1 ml 1 mg/m thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin.
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce interactor protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. After 1 h, 2.5 h and 4 h of protein overexpression, if you wish to do in cell analysis, remove 100 ml of cell culture. Centrifuge the cells and wash them twice with 40–50 ml of 10 mM potassium phosphate buffer (pH 7.0). Store the cell pellets at –80 °C for subsequent NMR analysis. To avoid cell lysis, do not flash freeze the cells. At each time point, remove 1 ml of the induced cells for analysis by SDS-PAGE; process the cells as described in Step 6. The OD600 of the induced interactor culture typically increases by less than 30% over 3–4 h of induction. ▲ CRITICAL STEP LB medium can be used instead of M9 for the IPTG induction; however, this usually results in a substantial increase in the OD600, which effectively dilutes the concentration of labeled target in the cells.
11. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    
    1. lowering temperature after induction and expressing for a longer period
    2. inducing in several stages, seperated by 2-3 h.
       
12. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Casamino acids
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl, MgCl₂, thiamine, glucose or glycerol
• Escherichia coli BL21 (DE3) (Novagen) or derivatives
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 g NaCl

M9 medium For 1 l, combine the following:

• 6 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 g NaCl

• 2 ml 1 M MgSO₄
• 1 ml 1 mg/m thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin.
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce interactor protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. After 1 h, 2.5 h and 4 h of protein overexpression, if you wish to do in cell analysis, remove 100 ml of cell culture. Centrifuge the cells and wash them twice with 40–50 ml of 10 mM potassium phosphate buffer (pH 7.0). Store the cell pellets at –80 °C for subsequent NMR analysis. To avoid cell lysis, do not flash freeze the cells. At each time point, remove 1 ml of the induced cells for analysis by SDS-PAGE; process the cells as described in Step 6. The OD600 of the induced interactor culture typically increases by less than 30% over 3–4 h of induction. ▲ CRITICAL STEP LB medium can be used instead of M9 for the IPTG induction; however, this usually results in a substantial increase in the OD600, which effectively dilutes the concentration of labeled target in the cells.
11. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    
    1. lowering temperature after induction and expressing for a longer period
    2. inducing in several stages, seperated by 2-3 h.
       
12. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Casamino acids
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)

• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

• 10 g peptone from casein
• 5 g yeast extract

M9 medium For 1 l, combine the following:

• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.

1. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
   
   1. lowering temperature after induction and expressing for a longer period
   2. inducing in several stages, seperated by 2-3 h.
      
2. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
   

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Casamino acids
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)

• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 gNaCl

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 MMgSO4
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce interactor protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. After 1 h, 2.5 h and 4 h of interactor protein overexpression, if you wish to do in cell analysis, remove 100 ml of cell culture. Centrifuge the cells and wash them twice with 40–50 ml of 10 mM potassium phosphate buffer (pH 7.0). Store the cell pellets at –80 °C for subsequent NMR analysis. To avoid cell lysis, do not flash freeze the cells. At each time point, remove 1 ml of the induced cells for analysis by SDS-PAGE; process the cells as described in Step 6. The OD600 of the induced interactor culture typically increases by less than 30% over 3–4 h of induction. ▲ CRITICAL STEP LB medium can be used instead of M9 for the IPTG induction; however, this usually results in a substantial increase in the OD600, which effectively dilutes the concentration of labeled target in the cells.
11. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    
    1. lowering temperature after induction and expressing for a longer period
    2. inducing in several stages, seperated by 2-3 h.
       
12. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Casamino acids
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)

• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 gNaCl

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 MMgSO4
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce interactor protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. After 1 h, 2.5 h and 4 h of interactor protein overexpression, if you wish to do in cell analysis, remove 100 ml of cell culture. Centrifuge the cells and wash them twice with 40–50 ml of 10 mM potassium phosphate buffer (pH 7.0). Store the cell pellets at –80 °C for subsequent NMR analysis. To avoid cell lysis, do not flash freeze the cells. At each time point, remove 1 ml of the induced cells for analysis by SDS-PAGE; process the cells as described in Step 6. The OD600 of the induced interactor culture typically increases by less than 30% over 3–4 h of induction. ▲ CRITICAL STEP LB medium can be used instead of M9 for the IPTG induction; however, this usually results in a substantial increase in the OD600, which effectively dilutes the concentration of labeled target in the cells.
11. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    
    1. lowering temperature after induction and expressing for a longer period
    2. inducing in several stages, seperated by 2-3 h.
       
12. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Casamino acids

• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 gNaCl

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 MMgSO4
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce interactor protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. After 1 h, 2.5 h and 4 h of interactor protein overexpression, if you wish to do in cell analysis, remove 100 ml of cell culture. Centrifuge the cells and wash them twice with 40–50 ml of 10 mM potassium phosphate buffer (pH 7.0). Store the cell pellets at –80 °C for subsequent NMR analysis. To avoid cell lysis, do not flash freeze the cells. At each time point, remove 1 ml of the induced cells for analysis by SDS-PAGE; process the cells as described in Step 6. The OD600 of the induced interactor culture typically increases by less than 30% over 3–4 h of induction. ▲ CRITICAL STEP LB medium can be used instead of M9 for the IPTG induction; however, this usually results in a substantial increase in the OD600, which effectively dilutes the concentration of labeled target in the cells.
11. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    
    1. lowering temperature after induction and expressing for a longer period
    2. inducing in several stages, seperated by 2-3 h.
       
12. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

LB medium and plates For 1 l, combine the following:

M9 medium For 1 l, combine the following:

MATERIALS

REAGENTS

• Ampicillin or carbenicillin

• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 g NaCl

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 g NaCl

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 M MgSO4
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with the pBADHisA and/or pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin and 35 mg l–1 kanamycin (LB-Ap-Kn). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter (PBAD).if used. Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg l–1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin and 35 mg l–1 kanamycin.
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce target protein overexpression by adding 1% culture volume of 20% (wt/vol) L-arabinose.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.

MATERIALS

REAGENTS

• Ampicillin or carbenicillin * Casamino acids * Plasmid pRSF.1b (Novagen), containing the cDNA for the protein * M9 medium (see REAGENT SETUP) * M9 salts: M9 medium without the addition of NH₄Cl, MgCl₂, thiamine, of interest glucose or glycerol
• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 g NaCl

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4

• 0.7 g NH4Cl or [U-15N]NH4Cl

• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with the pBADHisA and/or pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin and 35 mg l–1 kanamycin (LB-Ap-Kn). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter (PBAD).if used. Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg l–1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin and 35 mg l–1 kanamycin.
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce target protein overexpression by adding 1% culture volume of 20% (wt/vol) L-arabinose.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.

MATERIALS

REAGENTS

• Ampicillin or carbenicillin * Casamino acids * Plasmid pRSF.1b (Novagen), containing the cDNA for the protein * M9 medium (see REAGENT SETUP) * M9 salts: M9 medium without the addition of NH₄Cl, MgCl₂, thiamine, of interest glucose or glycerol
• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 g NaCl^?

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 M MgSO4^?
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with the pBADHisA and/or pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin and 35 mg l–1 kanamycin (LB-Ap-Kn). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter (PBAD).if used. Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg l–1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin and 35 mg l–1 kanamycin.
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce target protein overexpression by adding 1% culture volume of 20% (wt/vol) L-arabinose.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.

MATERIALS

REAGENTS

• Ampicillin or carbenicillin * Casamino acids * Plasmid pRSF.1b (Novagen), containing the cDNA for the protein * M9 medium (see REAGENT SETUP) * M9 salts: M9 medium without the addition of NH₄Cl, MgCl₂, thiamine, of interest glucose or glycerol
• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG

• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 g NaCl^?

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 g NaCl^?

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 M MgSO4^?
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with the pBADHisA and/or pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin and 35 mg l–1 kanamycin (LB-Ap-Kn). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter (PBAD).if used. Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg l–1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin and 35 mg l–1 kanamycin.
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce target protein overexpression by adding 1% culture volume of 20% (wt/vol) L-arabinose.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.

MATERIALS

REAGENTS

• Ampicillin or carbenicillin * Casamino acids * Plasmid pRSF.1b (Novagen), containing the cDNA for the protein * M9 medium (see REAGENT SETUP) * M9 salts: M9 medium without the addition of NH₄Cl, MgCl₂, thiamine, of interest glucose or glycerol
• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG * LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 g NaCl^?

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 g NaCl^?

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 M MgSO4^?
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with the pBADHisA and/or pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin and 35 mg l–1 kanamycin (LB-Ap-Kn). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter (PBAD).if used. Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg l–1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin and 35 mg l–1 kanamycin.
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce target protein overexpression by adding 1% culture volume of 20% (wt/vol) L-arabinose.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.

MATERIALS

REAGENTS

• Ampicillin or carbenicillin * Casamino acids * Plasmid pRSF.1b (Novagen), containing the cDNA for the protein * M9 medium (see REAGENT SETUP)

• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG * LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 g NaCl^?

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 g NaCl^?

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 M MgSO4^?
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with the pBADHisA and/or pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin and 35 mg l–1 kanamycin (LB-Ap-Kn). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter (PBAD).if used. Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg l–1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin and 35 mg l–1 kanamycin.
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce target protein overexpression by adding 1% culture volume of 20% (wt/vol) L-arabinose.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.

MATERIALS

REAGENTS

• Ampicillin or carbenicillin * Casamino acids * Plasmid pRSF.1b (Novagen), containing the cDNA for the protein * M9 medium (see REAGENT SETUP)

• Escherichia coli BL21 (DE3) (Novagen) or derivatives (see REAGENT
• 0.5 M IPTG * LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 g NaCl^?

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 g NaCl^?

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 M MgSO4^?
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with the pBADHisA and/or pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin and 35 mg l–1 kanamycin (LB-Ap-Kn). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter (PBAD).if used. Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg l–1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin and 35 mg l–1 kanamycin.
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce target protein overexpression by adding 1% culture volume of 20% (wt/vol) L-arabinose.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.

MATERIALS

REAGENTS

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 g NaCl^?

M9 medium For 1 l, combine the following:

• 6 g Na2HPO4
• 3 g KH2PO4
• 0.5 g NaCl^?

• 0.7 g NH4Cl or [U-15N]NH4Cl
• 2 ml 1 M MgSO4^?
• 1 ml 1 mg ml–1 thiamine HCl
• 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol

PROCEDURE :

1. | Transform E. coli strain BL21 (DE3), or its derivatives, with the pBADHisA and/or pRSF.1b expression plasmid(s)
2. | Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin and 35 mg l–1 kanamycin (LB-Ap-Kn). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter (PBAD).if used. Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. | Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg l–1) instead of ampicillin.
4. | Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. | Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin and 35 mg l–1 kanamycin.
6. | Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. | Incubate the culture at 37 °C for 10 min and induce target protein overexpression by adding 1% culture volume of 20% (wt/vol) L-arabinose.
8. | Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.

LB medium and plates For 1 l, combine the following:

REAGENT Setup E. coli BL21 E. coli strain BL21 (DE3) lacks Lon protease and is well suited for overexpressing heterologous proteins. If the protein that is being expressed is of eukaryotic origin, then E. coli Rosetta (DE3) cells are recommended; this strain contains the pRARE plasmid, which has a P15A origin and confers chloramphenicol resistance, both of which are compatible with the expression plasmids used in this procedure.

LB medium and plates For 1 l, combine the following: 10 g peptone from casein 5 g yeast extract 5 g NaCl^? Autoclave and store up to 1 month at 4 °C. For LB plates, add 15 g l–1 agar before autoclaving. Autoclave and then cool to 50 °C before adding appropriate antibiotics. Pour 10-cm plates. Store plates up to 1 month at 4 °C.

M9 medium For 1 l, combine the following: 6 g Na2HPO4 3 g KH2PO4 0.5 g NaCl^? Adjust pH to 7–7.4 and add 10 μl 1 M CaCl2^?. Autoclave and store up to 1 month at 4 °C. Autoclaving after adding CaCl2^? will ensure that the calcium phosphate that precipitates will be solubilized. Before use, add the following sterile reagents: 0.7 g NH4Cl or [U-15N]NH4Cl 2 ml 1 M MgSO4^? 1 ml 1 mg ml–1 thiamine HCl 10 ml 20% (wt/vol) D-glucose or 4 ml glycerol Glucose suppresses transcription from the PBAD promoter. Therefore, to maintain full promoter activity when expressing protein from pBAD, glycerol should be used as a carbon source instead of glucose.

PROCEDURE : 1 | Transform E. coli strain BL21 (DE3), or its derivatives, with the pBADHisA and/or pRSF.1b expression plasmid(s) 2| Grow the transformed cells on an LB plate containing 100 mg l–1 ampicillin and 35 mg l–1 kanamycin (LB-Ap-Kn).

▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter (PBAD).if used. Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.

3| Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD600 of ~1.6.

▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg l–1) instead of ampicillin.

4| Centrifuge (~5000g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l. 5| Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing [U-15N]NH4Cl (0.7 g l–1) and glycerol (4 ml l–1) as the sole nitrogen and carbon sources, respectively, and 100 mg l–1 ampicillin and 35 mg l–1 kanamycin. 6| Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.

7| Incubate the culture at 37 °C for 10 min and induce target protein overexpression by adding 1% culture volume of 20% (wt/vol) L-arabinose.

8| Allow the production of labeled target protein to proceed for 1 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.