SDS PAGE

- Protein crystallography

Main steps:

   - Protein purification
     - Introduction
     - Step-by-Step
     - Common sense
     - Protocols
       - Stock solutions
       - Cell disruption
       - Protamin sulphate treatment
       - Analytical AM cut
       - Preparative AM cut
       - Protein precipitation by AM
       - Protein recovery from AM
       - Solubility of expression
       - Solubility of expression
       - Protein concentration
       - Protein concentration
     - Charts & Tables
     - Appendix
   - Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

Protocols and tips in protein purification or How to purify protein in one day.

Sveta's easy protocol

Stock solutions:

1.5 M tris-HCl, pH 8.8
1.0 M tris-HCl, pH 6.8
Acrylamide 30% : (30g acrylamide, 0.8g bisAA).Use readymade solution.
AA 20% (30% diluted 1.5 fold with H2O)
Persulphate ammonium 10% (freshly made)
SDS 10%
TEMED

10 X Running Buffer:
144 g Glycine, 30g tris, 10g SDS. For a run dilute 10 fold with distilled water. pH is 8.3, do not adjust !!!

Sample buffer:
for 20 ml of 5X buffer: 6.25 ml 1M tris pH 6.8, 2g SDS, 5g sucrose, 1mg bromphenol blue, water to 18 ml. Before use, add 0.1 ml of 0.5M DTT to 0.9 ml of buffer for reducing conditions.
It is better to use ready made 4x sample buffer and 10x reducing agent from Invitrogen.

Gel preparation

For separation gel (15%):

1.5M tris pH 8.8	¼ V
AA 30%	½ V
H₂O	¼ V
SDS 10%	1/100 V
PSA 10%	5 µl/ml
TEMED	1 - 2 µl/ml

For example, for 8ml of solution take 2ml tris pH 8.8, 2ml H₂O, 4ml AA, add 80µl 10% SDS, 10µl TEMED and 40µl PSA.

for gel (10%) take ½V 20% AA,
for gel (12.5%) - ¼V 30% + ¼V 20%,
for gel (7.5%) - ¼V 20% + ½ V H₂O.

Staking gel (5%):

1M tris pH 6.8	1/8 V
AA 20%	¼ V
H₂O	3/8 V
SDS 10%	1/100 V
PSA and TEMED as before

For example, for 4ml take 0.5ml buffer, 1 ml AA 20%, 2.5ml H₂O, 40µl SDS, 5µl TEMED, 20µl PSA.