ISOELECTRIC FOCUSSING OF MEMBRANE PROTEINS BY SLAB GEL METHOD

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:

  1. Prepare the gel plates as usual.
  2. 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.
  3. 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.
  4. 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.

    5.  
    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
  5. apply samples to gel and electrophorese at 400 V at least 20 h. (minimum of 8000 V-h).
  6. 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

  1. cut out gel strips about 1 cm wide
  2. soak gel strip for 30 min in a petri dish in regular electrophoresis buffer (Tris, glycine) with the addition of 4% SDS
  3. 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.
  4. 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.

Updated 01 December 2000.