Protein(65KDa) was purified by gst affinity chromatography by using GLUTATHIONE SEPHAROSE 4B resin.

these are my SDS - page conditions:

(2) 12 % separating gel

1.5M tris HCl pH 8.8 :4 ml                                  1.5M tris Hcl pH 6.8 : 4ml

Acrylamide/bisacrylamide 30% :6.2ml                 Acrylamide/bisacrylamide :6.2ml

10% SDS :160 ul                                                          10% SDS ; 160ul

10%  APS : 160 ul                                                        10 % APS : 160 ul

TEMED     : 16ul                                                               TEMED :16 ul

1X Running buffer (1litre)

3g of tris base( 0.1M)

14.4g glycine( 0.5M )

1g SDS (0.1%)

1x staining solution

100 ml acetic acid

400 ml metahanol

1g coomassie blue R-250, made upto 1l with distilled water.

1X destaining solution

100 ml acetic acid

200 ml methanol

made upto 1l with distilled water.

equal protein concentration of 10 ug/ml of samples are loaded into each well.Before loading sample was mixed with 2x lamelli  sample loading buffer boiled at 95 degree celsius for 5 min in thermocycle.

can you give some suggestions to improve the gel visibility.