band on western where no protein

Jim Hutchins hutchins at fiona.umsmed.edu
Thu Jun 1 21:07:39 EST 1995


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.

The problem seems to be much worse when using reducing agents, especially
beta-mercaptoethanol.  If a reducing agent is not absolutely essential, omit
it and see if that helps.  If it is essential, try dithiothreitol or 
iodoacetamide.  I don't know if these will eliminate the problem or not, but
it might be worth a try if you are desperate.

I would jokingly wager that this particular artifact is the major cause of
this horrifying tendency to the 'band cut out of a gel' type of figure, which
is one gel lane wide and 2 mm high and shows only a region of the blot from
(say) 100-105 kD when the band of interest is found at 102. :-)  But as the
graduate students know, this is a pet peeve of mine. :-)

Good luck.
--
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_



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