Blotting of proteins onto Immobilon membranes. In situ characterization and comparison with high-performance liquid chromatography.
The electrophoretic transfer from polyacrylamide gels to Immobilon [poly(vinylidene difluoride)] membranes of various proteins differing in molecular masses from 14,000 to 200,000 was performed, using both a semi-dry blotting apparatus and a standard blotting chamber. The blotted proteins were analyzed and sequenced with and without staining, and the initial yields of the degradation were examined. Furthermore, protein purification by blotting after one- and two-dimensional gel electrophoresis was compared with conventional HPLC methods. Optimum blotting conditions for in situ enzymatic or chemical cleavages of the proteins on the blots are described, and for the in situ hydrolysis followed by amino acid analysis and cysteine determination.
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, (Dahlem), F.R.G.
T
T
Choli
U
U
Kapp
B
B
Wittmann-Liebold
D015153:Blotting, Western / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D003545:Cysteine / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D009019:Morphinans; D010455:Peptides / Q000032:analysis; D011145:Polyvinyls; D011506:Proteins / Q000302:isolation & purification*; D005465:Fluorocarbon Polymers
D003488:Cyanogen Bromide; D003545:Cysteine; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D009019:Morphinans; D010455:Peptides; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers