Western problem

Jayakumar, R R.Jayakumar at RoswellPark.org
Wed Dec 31 09:25:30 EST 2003

> > >---
> > 
> > I was wondering about a couple of things.
> > 
> > Why use 4X Laemmli buffer?
Oh.. I might have failed to mention. The protein was prepared in 4 X Laemmli buffer and then finally diluted to the required volume so that the final concentration of Laemmli buffer is 1X.
> > 
> > Does your 'loading buffer' contain SDS?
Of course..... as SDS-PAGE is always done..
> > 
> > The problems could be due to bad transfer to the membrane too.
That is the problem I am trying to circumvent.  Most wire electrodes have a inherest problem of non-uniformity of electric field.  Plate electrodes normally have much more uniformity.  

> > 
> > Sorry, I couldn't help.
any help is appreciated.  Atleast you put in your efforts which I really appreciate.  If you have any other tips about improving uniformity of transfer across blots.. let me know.
    After I posted that question some time back,  I actually did manage to reduce the non-uniformity of transfer to a large extent just by adding 0.05 % SDS in the transfer buffer and running at reduced volts at room temperature. But I still get a slightly (20-30 %) lower transfer towards the center of the blot.  Literature tells me that it is quite normal in westerns and people use plate electrodes to overcome that.  If you have any other idea.. let me know.  I use Immobilon P PVDF membranes (0.45 u) in a Mini-proteanII BioRad western aparatus.
> > 
> > Rolands G. Aravindan
> > rolands_aravindan at yahoo.com
> > hi,
> if the loading buffer contains SDS then you dont need to resuspend the
> pellet.After boiling all membrane proteins would get solubilised in
> the regular loading buffer.inconsistancies could have arisen because
> of the particulate matter in the sample.
I have been spinning down the protein sample everytime after that.  But still the same problem.  I can assure you it is not the particulate matter.  Thanks anyway for the thought.  


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