Ⅰ.Sensitivity Comparison of Different Protein Staining Methods.

Ⅱ.Coomassie Blue Staining.
CBB staining solution
0.5% Coomassie brilliant blue G250 or R250
40% methanol   10% acetic acid
Dissolve CBB in methanol and stir continuously for 15min.Acetic acid addition and ddH₂o.
Decolorizing solution
30% methanol   10% acetic acid
Staining method
1.Dye on a shaker for 30 minutes.
2.Decolorization to protein point or band.
3.ddH₂o Wash 3-5 times.

Ⅲ .Colloidal Coomassie Staining （Cambridge centre for porteomics）
sensitivity = ~100ng
1.Fixed:methanol/acetic acid/H₂o(45:1:54),at least 20min.
2.Dyeing 12-18hr.
Dyeing solution:17% (w/v) ammonium sulphate  34% methanol  0.5% acetic acid  0.1% (w/v) Coomassie G250
3.Decolorization:Decolorize with H₂o until the protein point and background are clear.

Extraction and preservation of protein spots in two-dimensional electrophoresis

1.Using PDQuest software or naked eye comparison, find out the protein points of interest, and make marks and records.
2.Rinse the glue with milliQ water twice.
3.Flush Ep tube with chromatographically pure methanol and milliQ water.
4.Cut off the lower end of the gun head(200 µl) so that its inner diameter is slightly smaller than the diameter of protein spots. Use chromatographic pure methanol and MilliQ water rinse gun head.
5.Focus on the center of the spot and carefully cut off the protein,put in Ep tube,MilliQ water rinse twice.If the rubber block is too large, cut it into 1 x 1 mm film.
6.Mark and record the cut points,Store at -80°C or -20°C after freeze-drying.