A new polyacrylamide gel electrophoresis system. Separation of small peptides and proteins in a volatile buffer system after modification with a strongly acidic fluorescent NH2 reagent.
Polyacrylamide gel electrophoresis is one of the most efficient methods for separation and identification of proteins. A new gel electrophoresis system has been developed in order to facilitate both separation and extraction of peptides and proteins ranging in molecular weight from approximately 200 up to 100 000. This system involves the use of a volatile buffer, triethylamine/formic acid pH 11.7, and a reaction with a covalently binding NH2 reagent, 1,3,6-trisulfonylpyrene 8-isothiocyanate. Under these conditions, the addition of strongly negative charges to proteins is achieved which allows migration according to molecular weight. The modified proteins being fluorescent require neither fixation nor staining for their detection. This is an absolute requirement especially when dealing with small peptides. An additional advantage of such a modification is the increased solubility of the proteins which makes their extraction easier and more efficient. THe extracted proteins are easily freed from salts and other small molecules simply by evaporation and they are readily available for sequencing analysis using Edman degradation, carboxypeptidase digestion or amino acid analysis.
D000588:Amines; D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D017879:Isothiocyanates; D010455:Peptides / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D013861:Thiocyanates; D014835:Volatilization
D000588:Amines; D002021:Buffers; D007202:Indicators and Reagents; D017879:Isothiocyanates; D010455:Peptides; D011506:Proteins; D013861:Thiocyanates; C034660:1,3,6-trisulfonylpyrene 8-isothiocyanate