HELP! Hydrophobic/basic protein transfer

Arle Kruckeberg arle at biovax.leeds.ac.uk
Tue May 30 07:34:19 EST 1995


In article <3pvqfm$1g2 at crcnis3.unl.edu>
lzhang at unlinfo.unl.edu (lingyu zhang) writes:

> I will deeply appreciate any suggestions/comments on how to successful
> transfer hydrophobic and basic proteins for western blotting.
> Lingyu

I had trouble with Western transfer of yeast integral membrane proteins
using Towbin buffer and semi-dry transfer. At the suggestion of
colleagues I switched 
(1) to wet transfer (BioRad TransBlot-style device), with 30 volt
transfers in the cold overnight
and (2) to a modification of Towbin, namely 48 mM Tris/39 mM glycine,
0.05% SDS, 5% methanol. 
No protein left in the gel upon Coomassie staining, lots on the PVDF
filter by India Ink staining (0.01% in PBS/0.5% Tween20), no
punch-through (ie no India-ink-binding material on a second filter
behind the first), even with 1.5 mm thick gels.
Good luck!
Arle
*************************************************
a.k.a. Dr. Arthur L. Kruckeberg
 Department of Biochemistry and Molecular Biology
University of Leeds        phone  +44 +113 2333172
Leeds LS2 9JT              FAX    +44 +113 2333167
Great Britain              arle at biovax.leeds.ac.uk
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