Smeared Western Samples

Patrick bier13 at mail.com
Sat Nov 24 08:01:02 EST 2001


Sophie <sweiss at icr.ac.uk> wrote in message news:<20011109105429.9175.qmail at ww02.jatek.com>...
> Hello,
> 
> I am trying to run western blots to determine the level of protein
> expression in salmonella. I lyse the bacteria with 1% triton, 10% glycerol
> and 15 aprotonin. My protein is 46000Da and i run it on a 12% SDS PAGE gel.
> The markers are always nicely separated and transfer to the membrane.
> 
> My problem is that all my samples end up as horrible smears down the gel
> instead of compact bands. I thought it was a problem with the bacteria but
> the same thing happens when i use a highly purified version of my protein.
> Has anyone got any suggestions for why this is happening and how i can fix it.
> 
> thanks,
> 
> Sophie.
> 
> Hello Sophie,
 I am always using lymphoblast cells in my studie. And i lysate them
with NP-40 compined with protease inhibitors (aprotinine, leupeptine,
pepstatine and pefabloc) to inhibit protein degradation. I always make
the buffer fresh. I put the buffer to the cells, incubate ten minutes
and pull the lysate ten times through needle, then i centrifuge 10'
14000 rpm 4 at 4 degrees. Put sample buffer ( with B-
mercaptoethanol)at my lysate, boil 5 minutes and load the gel.
Hope you can do something with this and want to hear if you find the
solution to your problem

Patrick
> 
> <http://www.biowww.net/forum/read.php?f=6&i=817&t=817>




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