Difference: CatalaseCrystals (13 vs. 14)

Revision 1430 Nov 2007 - Main.MichaelStaropoli

 
META TOPICPARENT name="LabProtocols"
Contents

Catalase Crystals

Recrystallization Protocol

You will need

  • Catalase from Bovine liver - Vendor: Sigma CAS Number: 9001-05-2
  • Dialysis buttons or slides
  • 0.02M Phosphate buffer 6.3 pH
  • 10 % Sodium Chloride

Procedure

  1. Extract 1 mL of protein sample from the bottle using a syringe and put the sample in a microfuge tube.
  2. Centrifuge the sample at 4 degrees C and 13,000rpm for 10 minutes.
  3. Repeat these steps 4 times:
    • Remove and discard the supernatant.
    • Dissolve the pellet in distilled water and centrifuge at 4 degrees C and 13,000rpm for 10 minutes.
  4. Remove and discard the supernatant.
  5. Add drops of 10% Sodium Chloride solution to the pellet until it dissolves completely.
  6. Centrifuge the sample at 4 degrees C and 14,000rpm for 20 minutes.
  7. Transfer the supernatant to dialysis apparatus.
  8. Dialyse against a 0.02M pH 6.3 phosphate buffer solution for 24 hours.
  9. Remove the sample and mount on a grid with carbon support film.
  10. Negative stain with 2% UA.

* Figure 1 – This is the crystal lattice observed in crystallized catalase proteins. A good crystal is well ordered and has no dark streaks from contamination. Crystals that appear bent at lower magnification typically do not exhibit a well ordered lattice. (300,000X) (300kX):
300kx_lattice.jpg

Changed:
<
<		 * catalase crystal (20kX):
>
>		 * Figure 2 – This crystal is relatively isolated from free protein particles and was easily recognizable by its sharp edges and pronounced darker color. From low magnification, crystals show as a small splinter-like rod and are typically alone on a grid square. Unfortunately, the crystal is most likely multi-layered, meaning that there are two or more sheets of protein overlapping. The lattice on this crystal was well defined and well ordered. (20,000X)(20kX):
  20kx_whole.jpg
Changed:
<
<		 * 2D catalse crystal (15kX):
>
>		 * Figure 3 – This crystal appeared to have a two dimensional array of protein, which is ideal if the catalase is being used for calibration. The background in the sample shows some evidence of free protein, but it is not in a great enough quantity to obscure the crystal. At higher magnification, this crystal showed a well-ordered lattice. (15,000X):
  15kx_whole.jpg

-- KdDerr - 29 Nov 2007

  • Set ALLOWTOPICVIEW =

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