Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.
Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.
Warlow
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RS
Psychology Department, La Trobe University, Bundoora, Victoria, Australia.
Bernard
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CC
D000641:Ammonia; D003102:Colloids; D004591:Electrophoresis, Polyacrylamide Gel; D005404:Fixatives; D006801:Humans; D008214:Lymphocytes / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D008970:Molecular Weight; D012394:Rosaniline Dyes; D012834:Silver; D012967:Sodium Dodecyl Sulfate; D012997:Solvents; D013194:Staining and Labeling
D003102:Colloids; D005404:Fixatives; D008565:Membrane Proteins; D012394:Rosaniline Dyes; D012997:Solvents; D012967:Sodium Dodecyl Sulfate; D012834:Silver; D000641:Ammonia; C004692:coomassie Brilliant Blue