band on western where no protein

Tracy Aquilla aquilla at salus.med.uvm.edu
Tue Jun 6 07:04:22 EST 1995


In Article <3qlrpb$a1c at fiona.umsmed.edu>, hutchins at fiona.umsmed.edu (Jim
Hutchins) wrote:
>STRUCTURAL NEUROBIOLOGY LAB (laurette at msvax.mssm.edu) wrote:
>: Dear Methodologists-
>: I have run westerns for a while and in the last month
>: have a band 60-70 kD where there is no sample.
>: i wear gloves, block with milk and BSA [no change
>: from before] overnight at 4C and use components
>: like 2nd antibody from the vectastain kit [and DAB] to stain.
>
>: please help, my thesis is due soon!!!!
>
>: thanks,
>
>: laurette
>
>This is a terribly common problem with all Western blotting procedures.  The
>band is consistently found at 67 kD.
>
>Some say it is keratin.  If it is, maybe someone else has used the same
>gel plates, comb, etc. without being as careful as you.  You could acid wash
>everything that can be acid-washed.
[snip]
>--
>Jim Hutchins
>Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr
>http://fiona.umsmed.edu/~hutchins/  ***  mailto://hutchins@fiona.umsmed.edu
>``There are two tragedies in life: one is not getting what you want.
>The other is getting it.''  Oscar Wilde, _The Importance of Being Earnest_

BSA is about 67-68 kDa. If it has gotten into your protein samples at some
stage it may cross-react on the Western. This happens a lot.
    tracy



More information about the Methods mailing list