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

Link to Article in PubMed

PubMed ID

2440344

Journal Title

Analytical biochemistry