Simultaneous isolation of DNA, RNA, and antigenic protein exhibiting kinase activity from small tumor samples using guanidine isothiocyanate.
Correlative studies of genes and their expression in human tumors are often hampered by the small sample size and the need to use differing and incompatible techniques to obtain DNA, RNA, and protein. We describe an extension of the established guanidine isothiocyanate method for isolation of DNA and RNA which allows the simultaneous isolation of total cellular protein. The protein obtained by this method (from solid tumors and cell lines) was comparable to protein extracted by a standard detergent solubilization method. Antigenicity was retained as demonstrated by Western blotting for epidermal growth factor receptor and actin and by immunoprecipitation of p53. Kinase activity was similar in proteins extracted by the two methods. It seems probable that most monomeric proteins can be obtained in a form suitable for Western analysis and immunoprecipitation and that these may also retain some functional activity.
Coombs
L M
LM
Marie Curie Research Institute, Oxted, Surrey, United Kingdom.
Pigott
D
D
Proctor
A
A
Eydmann
M
M
Denner
J
J
Knowles
M A
MA
D000199:Actins / Q000032:analysis; D000951:Antigens, Neoplasm / Q000302:isolation & purification; D015153:Blotting, Western; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D066246:ErbB Receptors / Q000032:analysis; D006146:Guanidines; D017879:Isothiocyanates; D008722:Methods; D009369:Neoplasms / Q000201:enzymology*; D011233:Precipitin Tests; D011494:Protein Kinases / Q000276:immunology / Q000302:isolation & purification*; D012334:RNA, Neoplasm / Q000302:isolation & purification*; D013861:Thiocyanates; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
D000199:Actins; D000951:Antigens, Neoplasm; D004273:DNA, Neoplasm; D006146:Guanidines; D017879:Isothiocyanates; D012334:RNA, Neoplasm; D013861:Thiocyanates; D016159:Tumor Suppressor Protein p53; C054435:guanidine isothiocyanate; D011494:Protein Kinases; D066246:ErbB Receptors