Direct amino acid analysis of proteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel.
The band of appropriate proteins (basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, interleukin 2, and human leukocyte interferon alpha A) on a polyvinylidene difluoride (PVDF) membrane, which was electroblotted from sodium dodecyl sulfate (SDS)-polyacrylamide gel and then stained with Coomassie blue R-250, was cut out and directly hydrolyzed in HCl in the presence of thioglycolic acid for amino acid analysis. The analytical values agreed with those expected with recoveries of 29-47%, except that the value for tryptophan was very low or scarcely detected. This method was applied to the identification of human growth hormone (hGH) in a partially purified preparation. The amino acid composition of the band corresponding to about 2 micrograms of hGH agreed with the theoretical values. These results indicate that the band on the PVDF membrane can be directly hydrolyzed for amino acid analysis and that the method can be used for partially purified proteins separated using SDS-polyacrylamide gel electrophoresis.
Nakagawa
S
S
Research and Development Division, Takeda Chemical Industries, Ltd., Osaka, Japan.
Fukuda
T
T
D000596:Amino Acids / Q000032:analysis*; D007611:Aprotinin / Q000032:analysis; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D013006:Growth Hormone / Q000032:analysis; D006868:Hydrolysis; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000032:analysis; D007376:Interleukin-2 / Q000032:analysis; D008567:Membranes, Artificial; D008722:Methods; D011506:Proteins / Q000032:analysis*; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000032:analysis
D000596:Amino Acids; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D007376:Interleukin-2; D008567:Membranes, Artificial; D011506:Proteins; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D013006:Growth Hormone; D007611:Aprotinin; D014360:Trypsin Inhibitor, Kunitz Soybean