Modifications of The Protein Immunoblotting Techniques

The authors made some improvments of the protein immunoblotting techniques during studies on the centromere proteins in sone protists.The main modifications reported in this paper are as followings.1.Separating gels were modified from a uniform polyacrylamide gel of 12% to linear gradient gels of 5%-12% (or 15%),stacking gel from 5% to 3%,and the separation of protein bands in the gel was much improved.2.Traching dye was changed from bromphenol blue to the pigments in Euglena gracilis SDS-PAGE sample,and the proper electrophoresis stop can be conveniently determined.3.Electrophoretic transfer was changed from 400 Ma,3.5 hurs to 200 mA.17 hurs;the cooling of transfer from air in refrigerator of 4 degree centigrades to flowing water bath,so that the transfer was cooled more successfully.4.The NC memberanes were washed thoroughly with double distilled water,then treated with with 0.1% Triton X-100-TBS after transfer,in order to remove SDS in the protein bands transferred.5.The incubation of antigen-antibody reactions were modified from RT or 37 degree centigrades,1-2 hurs to 4 degree centigrades,thus the backgroung of blotting was much reduced and the specificity of antigen-antibody reactions were much improved.