Title
A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins
UMMS Affiliation
Department of Cell Biology
Date
5-1-1987
Document Type
Article
Subjects
Animals; Buffers; Chlamydomonas; Collodion; Electrochemistry; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Weight; Peptides; Proteins; Staining and Labeling
Abstract
We have developed conditions for the efficient electrotransfer from polyacrylamide gels to nitrocellulose sheets of a broad size range of proteins (Mr 8,000 to Mr greater than 400,000). The important features of this procedure include a two-step electrotransfer, beginning with elution of low-molecular-weight polypeptides at a low current density (approximately 1 mA/cm2) for 1 h, followed by prolonged electrotransfer (16-20 h) at high current density (approximately 3.5-7.5 mA/cm2) in conditions that favor the elution of high-molecular-weight proteins. The transfer buffer includes 0.01% sodium dodecyl sulfate to enhance protein elution, and 20% methanol to improve the retention of proteins on the nitrocellulose sheet. The nitrocellulose is air-dried after transfer is complete to eliminate loss of proteins during subsequent processing. This transfer procedure works well with proteins prepared from many different cell types, and is suitable for use with all polyacrylamide gel systems tested. With little or no modification, our method should also be applicable to transfer membranes other than nitrocellulose.
Rights and Permissions
Citation: Anal Biochem. 1987 May 1;162(2):370-7.
Related Resources
PubMed ID
2440344
Journal Title
Analytical biochemistry