Home

About

Advanced Search

Browse by Discipline

Scientific Societies

E-print Alerts

Add E-prints

E-print Network
FAQHELPSITE MAPCONTACT US


  Advanced Search  

 
EFFICIENT ELECTROBLOTTlNG AND ON-MEMBRANE PROTEOLYSIS AT HIGH SENSITIVITY FOR SEQUENCE ANALYSIS
 

Summary: EFFICIENT ELECTROBLOTTlNG AND ON-MEMBRANE PROTEOLYSIS AT HIGH SENSITIVITY FOR
SEQUENCE ANALYSIS
TUTORIAL 5: ABRF'96: BIOMOLECULAR TECHNIQUES
David W. Speicher, Jacek Mozdzanowski, and David F. Reim
The Wistar Institute, Philadelphia, PA 19104
The most effective final purification step for isolating proteins at low picomole levels prior to sequence analysis is to
utilize either ID or 2D polyacrylamide gel electrophoresis (PAGE). These separated proteins can then be either digested
in the gel with a protease or the proteins can be electrotransfe-red onto a high retention PVDF membrane. One of the
advantages of electroblotting proteins onto PVDF membranes is that replicate lanes from a single gel can be used for N-
terminal sequencing, proteolysis/peptide separation/intemal sequencing and/or other analysis methods such as Western
analysis. In this tutorial, a high yield electroblotting method suitable for most proteins will be demonstrated and
representative results will be illustrated. This method uses high retention PVDF membranes such as ProBlott or Trans-
Blot, a 10 mM Tris, 100 mM Glycine, 10% methanol transfer buffer and a tank-type transfer unit with solid electrodes
(1). The single most important factor in efficient electroblotting is the amount of SDS present in the gel during
electrotransfer. High amounts of SDS interfere with protein binding to the PVDF membrane resulting in "blow through"
while low amounts of SDS or too rapid dissociation of SDS from the protein during the transfer will dramatically reduce
yields by leaving substantial amounts of the protein in the gel after transfer. Related factors that must be considered is the
gel thickness (optimum is 1. 0 - 1. 5 mm), the amount of SDS in the gel matrix and in the electrode buffer, and the
amount of methanol in the transfer buffer. Guidelines for identifying electrotransfer problems and ensuring optimal
electrotranfer yields will be presented together with strategies for optimizing yields of "difficult" proteins including very

  

Source: Andrews, Anne M. - Huck Institutes of the Life Sciences, Pennsylvania State University

 

Collections: Biology and Medicine