Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.
D000097:Acetonitriles; D003101:Collodion; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification*; D011725:Pyridines; D012997:Solvents; D013696:Temperature; D013997:Time Factors
D000097:Acetonitriles; D003902:Detergents; D006146:Guanidines; D011506:Proteins; D011725:Pyridines; D012997:Solvents; D003101:Collodion; D019791:Guanidine; C023666:pyridine; C032159:acetonitrile