Difference: MicroDialysisPlatesTool (1 vs. 12)

Micro-dialysis plates

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

Microdialysis plate overview

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Schematic top view

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Schematic cross-section view

Procedures for using XZ-96-D plates:

1. Pippette 0.5ml of reagent into the reagent wells.
2. Pippette 4~10ml of protein sample into the protein wells.
3. Centrifuge the plate at 1000rpm for 1min to get rid of any trapped bubbles.
4. Seal the plate.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).

Micro-dialysis plates

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

Microdialysis plate overview

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Schematic top view

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Schematic cross-section view

Procedures for using XZ-96-D plates:

1. Pippette 0.5ml of reagent into the reagent wells.
2. Pippette 4~10ml of protein sample into the protein wells.
3. Centrifuge the plate at 1000rpm for 1min to get rid of any trapped bubbles.
4. Seal the plate.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).

Micro-dialysis plates

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

Microdialysis plate overview

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Schematic top view

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Schematic cross-section view

Procedures for using XZ-96-D plates:

1. Pippette 0.5ml of reagent into the reagent wells.
2. Pippette 4~10ml of protein sample into the protein wells.
3. Centrifuge the plate at 1000rpm for 1min to get rid of any trapped bubbles.
4. Seal the plate.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).

Micro-dialysis plates

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

Microdialysis plate overview

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Schematic top view

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Schematic cross-section view

Procedures for using XZ-96-D plates:

1. Pippette 0.5ml of reagent into the reagent wells.
2. Pippette 4~10ml of protein sample into the protein wells.
3. Centrifuge the plate at 1000rpm for 1min to get rid of any trapped bubbles.
4. Seal the plate.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).

Micro-dialysis plates

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

Microdialysis plate overview

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Schematic top view

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Schematic cross-section view

Procedures for using XZ-96-D plates:

1. Pippette 0.5ml of reagent into the reagent wells.
2. Pippette 4~10ml of protein sample into the protein wells.
3. Centrifuge the plate at 1000rpm for 1min to get rid of any trapped bubbles.
4. Seal the plate.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

Microdialysis plate overview

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Schematic top view

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Schematic cross-section view

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).

Micro-dialysis plates experiments

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Performance

The performance of the minidialysis plate was evaluated by quantifying the rate of detergent removal through thin layer chromatography (TLC) and by producing crystals of the membrane protein P2A3.

For the detergent removal tests, three detergents with very different CMC’s were evaluated: 100mg/ml OG (CMC: 5.3 mg/ml), 30mg/ml DM (CMC: 0.87mg/ml), and 20mg/ml DDM (CMC: 0.087mg/ml).

OG was rapidly eliminated from the dialysis wells, and after six buffer exchanges all OG had been removed. Interestingly, the rate of dialysis seems to be a function of time, and independent of buffer exchange frequency (compare figure 3a and 3b). Thus, the gradient difference over the dialysis membrane seems not to be the rate limiting factor for OG removal from the sample well, since frequent buffer exchanges do not increase that rate. In fact, the rate of removal is essentially linear with time for the first 40h of dialysis or until approximately 80% of the detergent has been removed, whereafter the rate slows down considerably. These results indicate that the rate of dialysis may be governed by some intrinsic properties of OG, such as its interaction with the dialysis membrane that may reduce its diffusion speed, or disassembly of the OG micelles.

DM was removed from the dialysis wells at an intermediate rate. After around 10 buffer exchanges or 150h, almost all of the detergent was removed. Note that also for DM the rate seems to be independent of the buffer exchange frequency, which is especially evident for buffer exchange no 7 where the buffer was left in the plate for close to 40h before it was changed, and therefore allowed a lot of DM to be removed at once.

Also in this case the there seems to be another rate-limiting step to the speed of removal than the frequency of buffer exchanges, since also here the initial part of the removal curve is essentially linear when plotted against time but not when plotted against buffer exchange number.

As expected, DDM was removed considerably slower than the other detergents and required roughly 25 buffer exchanges and a total dialysis time of two weeks.

These results are similar to the results obtained with the crystallization block (Vink et al., 2007) and confirm that even detergents with very low CMCs can be consistently removed through dialysis.

Next, the protein P2A3 (purified in DDM) was dialyzed in the minidialysis plates using conditions previously established with the larger dialysis block and with dialysis buttons. After two weeks of dialysis, with buffer exchanges twice per day, long tubular crystals were observed with a similar abundance to those obtained with the other dialysis devices (figure 4). These results clearly demonstrate the functionality of the microfluidic dialysis plate and work is currently underway to incorporate the plate into our crystallization pipeline.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).

Micro-dialysis plates experiments

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Performance

The performance of the minidialysis plate was evaluated by quantifying the rate of detergent removal through thin layer chromatography (TLC) and by producing crystals of the membrane protein P2A3.

For the detergent removal tests, three detergents with very different CMC’s were evaluated: 100mg/ml OG (CMC: 5.3 mg/ml), 30mg/ml DM (CMC: 0.87mg/ml), and 20mg/ml DDM (CMC: 0.087mg/ml).

OG was rapidly eliminated from the dialysis wells, and after six buffer exchanges all OG had been removed. Interestingly, the rate of dialysis seems to be a function of time, and independent of buffer exchange frequency (compare figure 3a and 3b). Thus, the gradient difference over the dialysis membrane seems not to be the rate limiting factor for OG removal from the sample well, since frequent buffer exchanges do not increase that rate. In fact, the rate of removal is essentially linear with time for the first 40h of dialysis or until approximately 80% of the detergent has been removed, whereafter the rate slows down considerably. These results indicate that the rate of dialysis may be governed by some intrinsic properties of OG, such as its interaction with the dialysis membrane that may reduce its diffusion speed, or disassembly of the OG micelles.

DM was removed from the dialysis wells at an intermediate rate. After around 10 buffer exchanges or 150h, almost all of the detergent was removed. Note that also for DM the rate seems to be independent of the buffer exchange frequency, which is especially evident for buffer exchange no 7 where the buffer was left in the plate for close to 40h before it was changed, and therefore allowed a lot of DM to be removed at once.

Also in this case the there seems to be another rate-limiting step to the speed of removal than the frequency of buffer exchanges, since also here the initial part of the removal curve is essentially linear when plotted against time but not when plotted against buffer exchange number.

As expected, DDM was removed considerably slower than the other detergents and required roughly 25 buffer exchanges and a total dialysis time of two weeks.

These results are similar to the results obtained with the crystallization block (Vink et al., 2007) and confirm that even detergents with very low CMCs can be consistently removed through dialysis.

Next, the protein P2A3 (purified in DDM) was dialyzed in the minidialysis plates using conditions previously established with the larger dialysis block and with dialysis buttons. After two weeks of dialysis, with buffer exchanges twice per day, long tubular crystals were observed with a similar abundance to those obtained with the other dialysis devices (figure 4). These results clearly demonstrate the functionality of the microfluidic dialysis plate and work is currently underway to incorporate the plate into our crystallization pipeline.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).

* Scheme_TopView.png:

Micro-dialysis plates experiments

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Performance

The performance of the minidialysis plate was evaluated by quantifying the rate of detergent removal through thin layer chromatography (TLC) and by producing crystals of the membrane protein P2A3.

For the detergent removal tests, three detergents with very different CMC’s were evaluated: 100mg/ml OG (CMC: 5.3 mg/ml), 30mg/ml DM (CMC: 0.87mg/ml), and 20mg/ml DDM (CMC: 0.087mg/ml).

OG was rapidly eliminated from the dialysis wells, and after six buffer exchanges all OG had been removed. Interestingly, the rate of dialysis seems to be a function of time, and independent of buffer exchange frequency (compare figure 3a and 3b). Thus, the gradient difference over the dialysis membrane seems not to be the rate limiting factor for OG removal from the sample well, since frequent buffer exchanges do not increase that rate. In fact, the rate of removal is essentially linear with time for the first 40h of dialysis or until approximately 80% of the detergent has been removed, whereafter the rate slows down considerably. These results indicate that the rate of dialysis may be governed by some intrinsic properties of OG, such as its interaction with the dialysis membrane that may reduce its diffusion speed, or disassembly of the OG micelles.

DM was removed from the dialysis wells at an intermediate rate. After around 10 buffer exchanges or 150h, almost all of the detergent was removed. Note that also for DM the rate seems to be independent of the buffer exchange frequency, which is especially evident for buffer exchange no 7 where the buffer was left in the plate for close to 40h before it was changed, and therefore allowed a lot of DM to be removed at once.

Also in this case the there seems to be another rate-limiting step to the speed of removal than the frequency of buffer exchanges, since also here the initial part of the removal curve is essentially linear when plotted against time but not when plotted against buffer exchange number.

As expected, DDM was removed considerably slower than the other detergents and required roughly 25 buffer exchanges and a total dialysis time of two weeks.

These results are similar to the results obtained with the crystallization block (Vink et al., 2007) and confirm that even detergents with very low CMCs can be consistently removed through dialysis.

Next, the protein P2A3 (purified in DDM) was dialyzed in the minidialysis plates using conditions previously established with the larger dialysis block and with dialysis buttons. After two weeks of dialysis, with buffer exchanges twice per day, long tubular crystals were observed with a similar abundance to those obtained with the other dialysis devices (figure 4). These results clearly demonstrate the functionality of the microfluidic dialysis plate and work is currently underway to incorporate the plate into our crystallization pipeline.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005). * Im_Plate.png:

* Scheme_CrossSection.png:

Micro-dialysis plates experiments

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Performance

The performance of the minidialysis plate was evaluated by quantifying the rate of detergent removal through thin layer chromatography (TLC) and by producing crystals of the membrane protein P2A3.

For the detergent removal tests, three detergents with very different CMC’s were evaluated: 100mg/ml OG (CMC: 5.3 mg/ml), 30mg/ml DM (CMC: 0.87mg/ml), and 20mg/ml DDM (CMC: 0.087mg/ml).

OG was rapidly eliminated from the dialysis wells, and after six buffer exchanges all OG had been removed. Interestingly, the rate of dialysis seems to be a function of time, and independent of buffer exchange frequency (compare figure 3a and 3b). Thus, the gradient difference over the dialysis membrane seems not to be the rate limiting factor for OG removal from the sample well, since frequent buffer exchanges do not increase that rate. In fact, the rate of removal is essentially linear with time for the first 40h of dialysis or until approximately 80% of the detergent has been removed, whereafter the rate slows down considerably. These results indicate that the rate of dialysis may be governed by some intrinsic properties of OG, such as its interaction with the dialysis membrane that may reduce its diffusion speed, or disassembly of the OG micelles.

DM was removed from the dialysis wells at an intermediate rate. After around 10 buffer exchanges or 150h, almost all of the detergent was removed. Note that also for DM the rate seems to be independent of the buffer exchange frequency, which is especially evident for buffer exchange no 7 where the buffer was left in the plate for close to 40h before it was changed, and therefore allowed a lot of DM to be removed at once.

Also in this case the there seems to be another rate-limiting step to the speed of removal than the frequency of buffer exchanges, since also here the initial part of the removal curve is essentially linear when plotted against time but not when plotted against buffer exchange number.

As expected, DDM was removed considerably slower than the other detergents and required roughly 25 buffer exchanges and a total dialysis time of two weeks.

These results are similar to the results obtained with the crystallization block (Vink et al., 2007) and confirm that even detergents with very low CMCs can be consistently removed through dialysis.

Next, the protein P2A3 (purified in DDM) was dialyzed in the minidialysis plates using conditions previously established with the larger dialysis block and with dialysis buttons. After two weeks of dialysis, with buffer exchanges twice per day, long tubular crystals were observed with a similar abundance to those obtained with the other dialysis devices (figure 4). These results clearly demonstrate the functionality of the microfluidic dialysis plate and work is currently underway to incorporate the plate into our crystallization pipeline.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005). * Im_Plate.png:

Micro-dialysis plates experiments

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Performance

The performance of the minidialysis plate was evaluated by quantifying the rate of detergent removal through thin layer chromatography (TLC) and by producing crystals of the membrane protein P2A3.

For the detergent removal tests, three detergents with very different CMC’s were evaluated: 100mg/ml OG (CMC: 5.3 mg/ml), 30mg/ml DM (CMC: 0.87mg/ml), and 20mg/ml DDM (CMC: 0.087mg/ml).

OG was rapidly eliminated from the dialysis wells, and after six buffer exchanges all OG had been removed. Interestingly, the rate of dialysis seems to be a function of time, and independent of buffer exchange frequency (compare figure 3a and 3b). Thus, the gradient difference over the dialysis membrane seems not to be the rate limiting factor for OG removal from the sample well, since frequent buffer exchanges do not increase that rate. In fact, the rate of removal is essentially linear with time for the first 40h of dialysis or until approximately 80% of the detergent has been removed, whereafter the rate slows down considerably. These results indicate that the rate of dialysis may be governed by some intrinsic properties of OG, such as its interaction with the dialysis membrane that may reduce its diffusion speed, or disassembly of the OG micelles.

DM was removed from the dialysis wells at an intermediate rate. After around 10 buffer exchanges or 150h, almost all of the detergent was removed. Note that also for DM the rate seems to be independent of the buffer exchange frequency, which is especially evident for buffer exchange no 7 where the buffer was left in the plate for close to 40h before it was changed, and therefore allowed a lot of DM to be removed at once.

Also in this case the there seems to be another rate-limiting step to the speed of removal than the frequency of buffer exchanges, since also here the initial part of the removal curve is essentially linear when plotted against time but not when plotted against buffer exchange number.

As expected, DDM was removed considerably slower than the other detergents and required roughly 25 buffer exchanges and a total dialysis time of two weeks.

These results are similar to the results obtained with the crystallization block (Vink et al., 2007) and confirm that even detergents with very low CMCs can be consistently removed through dialysis.

Next, the protein P2A3 (purified in DDM) was dialyzed in the minidialysis plates using conditions previously established with the larger dialysis block and with dialysis buttons. After two weeks of dialysis, with buffer exchanges twice per day, long tubular crystals were observed with a similar abundance to those obtained with the other dialysis devices (figure 4). These results clearly demonstrate the functionality of the microfluidic dialysis plate and work is currently underway to incorporate the plate into our crystallization pipeline.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).

Micro-dialysis plates experiments

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Performance

The performance of the minidialysis plate was evaluated by quantifying the rate of detergent removal through thin layer chromatography (TLC) and by producing crystals of the membrane protein P2A3.

For the detergent removal tests, three detergents with very different CMC’s were evaluated: 100mg/ml OG (CMC: 5.3 mg/ml), 30mg/ml DM (CMC: 0.87mg/ml), and 20mg/ml DDM (CMC: 0.087mg/ml).

OG was rapidly eliminated from the dialysis wells, and after six buffer exchanges all OG had been removed. Interestingly, the rate of dialysis seems to be a function of time, and independent of buffer exchange frequency (compare figure 3a and 3b). Thus, the gradient difference over the dialysis membrane seems not to be the rate limiting factor for OG removal from the sample well, since frequent buffer exchanges do not increase that rate. In fact, the rate of removal is essentially linear with time for the first 40h of dialysis or until approximately 80% of the detergent has been removed, whereafter the rate slows down considerably. These results indicate that the rate of dialysis may be governed by some intrinsic properties of OG, such as its interaction with the dialysis membrane that may reduce its diffusion speed, or disassembly of the OG micelles.

DM was removed from the dialysis wells at an intermediate rate. After around 10 buffer exchanges or 150h, almost all of the detergent was removed. Note that also for DM the rate seems to be independent of the buffer exchange frequency, which is especially evident for buffer exchange no 7 where the buffer was left in the plate for close to 40h before it was changed, and therefore allowed a lot of DM to be removed at once.

Also in this case the there seems to be another rate-limiting step to the speed of removal than the frequency of buffer exchanges, since also here the initial part of the removal curve is essentially linear when plotted against time but not when plotted against buffer exchange number.

As expected, DDM was removed considerably slower than the other detergents and required roughly 25 buffer exchanges and a total dialysis time of two weeks.

These results are similar to the results obtained with the crystallization block (Vink et al., 2007) and confirm that even detergents with very low CMCs can be consistently removed through dialysis.

Next, the protein P2A3 (purified in DDM) was dialyzed in the minidialysis plates using conditions previously established with the larger dialysis block and with dialysis buttons. After two weeks of dialysis, with buffer exchanges twice per day, long tubular crystals were observed with a similar abundance to those obtained with the other dialysis devices (figure 4). These results clearly demonstrate the functionality of the microfluidic dialysis plate and work is currently underway to incorporate the plate into our crystallization pipeline.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).