Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing.
A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.
Hsieh
J C
JC
Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Lin
F P
FP
Tam
M F
MF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration; D005898:Glass; D005982:Glutathione Transferase / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology; D011506:Proteins / Q000302:isolation & purification; D051381:Rats
D000596:Amino Acids; D011506:Proteins; D005982:Glutathione Transferase