Western problem

vivek tnvivek at mrdg.iisc.ernet.in
Wed Dec 31 07:37:17 EST 2003


"Aravind" <remove this-aravind at charter.net> wrote in message news:<vsk6crqs5t5k34 at corp.supernews.com>...
> Jayakumar wrote in message ...
> >Hi all..
> >    I am having a very strange problem with my western blots.  Here is what
> >I do.  I have these mitochondrial suspensions of known protein conc. (in a
> >buffer containing 70mM sucrose, 220mM mannitol, 2mM Hepes pH 7.44, 0.5mg/ml
> >BSA and other protease inhibitors).  I suspend my mito pellet by vortexing
> >(I am not bothered about its integrity) and then by triturating it with a
> >P-200 pipettman, so as to homogeneously suspend it.  I then go ahead and
> >prepare the sample with the required amount of protein with 4X Laemmli
> >buffer.  I have to load them in dulicates and in several concentrations.  I
> >make a master mix of the protein with the Laemmli buffer, boil it, spin it
> >briefly and vortex it (since I want to load everything including the
> >precipitates whcih may have membrane proteins).  Then I load the required
> >volume from this master mix so as to get the required concentrations in
> >duplicates.
> >     Strangely I dont get uniformity in band intensity between the same
> >concentrations.  Some bands dont appear at all, while some are very intense
> >even though all of them are loaded with the same volume that should have
>  the
> >same protein concentrations.
> >     I tried extracting the mito with a buffer containing NP-40 (IGEPAL)
>  and
> >then doing the same thing.  I am still getting non-uniform band intensity
> >(Some are light and some are dark), for the same protein loads.
> >     Please let me know what I am doing wrong here.  I am at my wits end.
> >     Any help would be gratefully acknowledged.
> >regards
> >Jai
> >
> >
> >---
> 
> I was wondering about a couple of things.
> 
> Why use 4X Laemmli buffer?
> 
> Does your 'loading buffer' contain SDS?
> 
> The problems could be due to bad transfer to the membrane too.
> 
> Sorry, I couldn't help.
> 
> 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.

vivek



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