REFERENCE: Ames, G.F.L. and Nikaido, H. 1976. Biochemistry. 15:616-623.
MATERIALS:
Gel solution:
| 1.05 g | acrylamide |
| 0.032 g | bis-acrylamide |
| 8.25 g | urea |
| 6.5 ml | distilled H2O |
| 0.3 ml | pH 4 - 6 ampholines |
| 0.3 ml | pH 6 - 8 ampholines |
| 0.15 ml | pH 3.5 - 10 ampholines |
| 0.03 ml | 10% ammonium persulphate |
| 1.5 ml | 20% TX-100 |
| 20 µl | TEMED |
Sample preparation:
| 25.0 µl | protein (2 to 10 mg/ml, depending on number of proteins in sample) |
| 9.0 µl | dH2O |
| 2.5 µl | 0.5M Tris-HCl, pH 6.8 |
| 1.1 µl | 2-mercaptoethanol |
| 4.5 µl | 10% SDS |
Heat for 5 minutes at 100oC and add:
| 2.5 µl | mix of 3 ampholines (same proportions as added to gel |
| 4.0 µl | 10% TX-100 |
| 25.0 mg | urea |
Top buffer: 0.02M NaOH (0.4 g/500 ml)\
Bottom buffer: 0.01M H3PO4 (0.57 ml/500 ml)
METHODS:
Prepare the gel plates as usual.
Mix all the gel solution components except the TEMED. Degas for exactly 2 minutes at exactly 20 psi on the old vacuum pump. Over-degassing has led to premature polymerisation in the past.
Add the TEMED and pour quickly into plates with the comb in place. NB: Polymerisation of these gels may often take up to an hour especially near the comb. Remove the comb carefully and don't give up too soon.
Prepare samples as above. The concentration of urea in the samples has been reduced slightly from 8M because it would often "precipitate" in the syringe.
| While preparing samples, pre-focus the gel as follows: place top and bottom buffers in chambers of gel attach leads and run with no samples as follows |
| 50 V Constant voltage 15 min |
| 200 V Constant voltage 15 min |
| 300 V Constant voltage 15 min |
| 400 V Constant voltage 30 min |
apply samples to gel and electrophorese at 400 V at least 20 h. (minimum of 8000 V-h).
Gel can either be stained or used in the second dimension (below).
STAINING
Fixer:
17.3 g sulphosalicyclic acid
57.5 g trichloroacetic acid
500 ml dH2O
Stain: regular Coomassie stain (MeOH, HOAc) and destain.
SECOND DIMENSION
cut out gel strips about 1 cm wide
soak gel strip for 30 min in a petri dish in regular electrophoresis buffer (Tris, glycine) with the addition of 4% SDS
the second dimension gel is a regular SDS-PAGE gel. It is easier to place the gel strip on the top of the gel if the notched plate has a bevelled edge.
apply gel strip to top of second dimension gel making sure the edges are in close contact. Seal the gel slice with agarose at the sides and across the top to hold it in place and electrophorese as usual for SDS-PAGE.
首页
