Smeared Western Samples

Araluen Freeman araluen_f at nospam.geocities.com
Mon Nov 12 07:55:25 EST 2001

"Sophie" <sweiss at icr.ac.uk> wrote in message
news:20011109105429.9175.qmail at ww02.jatek.com...

> 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
> 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

Hi Sophie,

I've used a Triton X-100 based lysis buffer to gently lyse mammalian cells
for SDS-PAGE.  I found that sonicating my lysates for 30 seconds total (40%
duty cycle, microtip limit) cleared up my bands by improving the degree of
lysis and reducing the sample viscosity (easier loading).  You can also
centrifuge your samples after lysis and sonication to remove cellular debris
that may be interfering in your gels, however that might interfere with the
harvest of your protein if it is a membrane associated protein.

I don't know how you would explain drastic smears of your purified protein
unless it is degraded.  Other points I would check would be:

-    Are the gels being electrophoresed at no more than 15 mA
     per gel connected in parallel to your power supply?
-    Are the samples boiled for 3 minutes before loading as
     per the typical Laemmli (1970) gel protocol?
-    Is there perhaps too much protein loaded per lane?  Try
     reducing the total protein loaded for better band resolution.
-    Is there any literature you can refer to for similar work on
      your protein or organism?

I'd be interested to hear what caused your problem, Sophie, when you've
ironed it out.  It's always the pesky little problems that catch you out,
isn't it?  ;-)

More information about the Proteins mailing list

Send comments to us at biosci-help [At] net.bio.net