Protein extraction and western blotting from methacarn-fixed tissue.
Polyacrylamide gel electrophoresis (PAGE) of proteins with subsequent western blotting has become a routine technique for the analysis of proteins from both cultured cells and fresh whole tissue. We have developed a method to extract proteins from methacarn-fixed tissue which renders them suitable for SDS-PAGE and western blotting. With a panel of antibodies to specific intermediate filaments, transforming growth factor-alpha (TGF-alpha), and albumin, immunohistochemistry was performed in parallel with western blotting on sections cut from methacarn-fixed samples of normal rat liver and liver from rats treated under a regime which induces oval cell proliferation. Immunohistochemistry enabled the determination of changes in tissue distribution and abundance of the target proteins, which was mirrored by the corresponding western blot data. This technique can be especially effective when used in conjunction with immunohistochemistry. Tissue samples are easy to prepare, avoiding the precautions which need to be taken when handling fresh tissue (Abstract: J Pathol 1994; 173S: No. 41).
Lalani
E N
EN
Department of Histopathology, Royal Postgraduate Medical School, London, U.K.
Golding
M
M
Hudson
M
M
Chieffi
G
G
Stamp
G
G
Anilkumar
T V
TV
Sarraf
C
C
Alison
M R
MR
D000085:Acetates; D019342:Acetic Acid; D000818:Animals; D015153:Blotting, Western; D002725:Chloroform; D004591:Electrophoresis, Polyacrylamide Gel; D005404:Fixatives; D007124:Immunoenzyme Techniques; D007633:Keratins / Q000032:analysis; D008297:Male; D000432:Methanol; D011506:Proteins / Q000032:analysis*; D051381:Rats; D016707:Tissue Fixation / Q000379:methods*; D014746:Vimentin / Q000032:analysis
D000085:Acetates; D005404:Fixatives; D011506:Proteins; D014746:Vimentin; C046544:methacarn; D007633:Keratins; D002725:Chloroform; D019342:Acetic Acid; D000432:Methanol