Western Blotting of Membrane Proteins in the presence of SDS

Marivonne Marivonne
Fri Apr 23 12:29:58 EST 1993


In article <01GXBS80C7B6003K5N at nic.the.net>
SHAUN%JASON.DECNET at relay.the.net ("Shaun D. Black") writes:
>Dear Netters,
>     I'd appreciate your experience as to electroblotting of membrane 
>proteins on PVDF (polyvinyldenedifluoride) membranes.  We've tried a
number 
>of conditions but would be curious as to what others have found optimal
for 
>membrane proteins from SDS-PAGE -> PVDF.
>
>     1. Buffers:  [50mM Tris / 380mM Glycine] vs [25mM Tris / 190mM Gly]
>                  (or others?)

I use 20mM Tris/150mM Glycine. It works perfect for my system (I use an
Idea Scientific GENIE electroblotter, plate electrodes)

>
>     2. SDS    :  0% vs 0.1% (or others?)

I use 0.05%. I was advised not to use 0.1%, as it closely approximates (if
not being equivalent to) that used for electroeluting proteins from gels,
and you can have problems with proteins going through the PVDF if too much
SDS is present. However, I did it without SDS and transfer wasn't nearly
as good. 

>
>     3. Methanol: 20% required?

I haven't really tried it without MeOH (I use 20% as well). I'm not sure,
but the hydrophobicity of PVDF may require MeOH in the buffer.

>
>     4. Voltage / Time:  Is there an optimum or do membrane proteins
simply
>                         require more time?

The type of electroblotter you use (wire electrodes vs. plates, tank vs.
semidry) as well as the strength of your transfer buffer play a role in
this, as well as the size range of the proteins in question (there may be
other factors, I'm sure...). I use 24V for 1 1/2 hrs., the last hour in
ice bath. (I haven't tried shortening the time, although I mean to, it's
just one of those things where you KNOW this is going to work, so fiddling
seems wasteful if you don't mind waiting..... there's ALWAYS something to
occupy your time in the lab! :) )
>
>     5. Is it important to blot right after SDS-PAGE or can gels be
soaked
>        in blotting buffer overnight before electrotransfer?

I always blot right away. There can be some diffusion of your proteins
while still in the gel, and so the resolution of the bands you end up
transfering may be affected.
 
>
>Thanks in advance.  Your thoughts are much appreciated.  Cheers.  -Shaun

Have fun with you Westerns! (I know I do! ;) )

Marivonne Rodriguez



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