western blotting - transferring a small MW protein

vetimmunol at hau.nic.in vetimmunol at hau.nic.in
Fri Mar 22 23:23:16 EST 2002


Dear John 
Try using PVDF 0.22um using current @ 0.8mA/cm2 of the membrane size. 
You have to work out the duration for transfer,but generally transfer 
for 45 minutes at above mentioned constant current work very nice. 
Good luck!

Devender Kr. Saini,
Research associate,
Immunodiagnosis Lab,
Dept. Veterinary Parasitology,
College of Veterinary Science,
CCS Haryana Agricultural University,Hisar,Haryana,
India-125004.


----- Original Message -----
From: john.hines at yale.edu
Date: Saturday, March 23, 2002 8:35 am
Subject: western blotting - transferring a small MW protein

> Hey everyone,
> 
>   This is the first time that I have ever tried to do a western for
> something so small.  My target protein is 10 kDa.  I'm used to larger
> targets (40 - 100 kDa).  I'm doing wet transfer in the standard
> Tris/Glycine/SDS/methanol buffer from out of Harlow and Lane.  Pure
> nitrocellulose with the .45 micron pore size.
> 
>    What I'm looking for are recommended transfer conditions.  How 
> manyvolts (or milliamps) for how long a time?  I don't want to 
> blow my tiny
> protein through my nitrocellulose, but at the same time I want to 
> get as
> much of it onto the membrane as possible.
> 
> 
>    Does anyone out there with experience in western blotting for 
> smallerproteins have a place to start?
> 
> 
> John
> 
> john.hines at yale.edu
> 
> 

---




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