ECL Western Blotting, need help

Roland Halil s635141 at uottawa.ca
Tue Aug 12 11:34:39 EST 1997


In article <19970808041901.AAA14713 at ladder02.news.aol.com>,
yankiwski at aol.com (Yankiwski) wrote:

> NaN3 seems to inhibit the activity of HRP coupled to my secondary
> antibody.    

Dear Victor, 
   NaN3 definitely kills the HRPso it should be made fresh each time. 
However, since you're only incubating for an hour or so, you don't really
need the azide.  All of your other solutions (ie. the primary antibody and
blocking solutions need azide if you're incubating for a long time).  I
have successfully used PVDF without any problems for a while now.  Try
using TBS with a bit of Tween 20 in it (TBST) when washing.  Also, you
should always have dry milk in your TBST even when washing in order to
continually block non-specific binding.

> When this is used in my Westerns I get
> several bands.  Background is not very high, indicating that my washes are
> quite good, but how can I eliminate this nonspecific binding?  

How old is your antibody?  Even though you can store and reuse a primary
antibody in milk+TBST w/ Azide for some time, it WILL break down after a
while and give you extra bands even when your blocking is well done and
your background is low.  
   You can also usually get by with using just a few mL of your primary and
secondary solutions by sliding your PVDF membrane (which is much sturdier
than nitrocellulose) into a hybridization tube and letting is turn gently.
As opposed to covering your membranes in 10-40mL of solution and committing
large quantities of your antibody to a solution.
   As for the PVDF and whether it works or not, Amersham recently had a bad
batch of PVDF and if it suddenly stopped working call the company and ask
them.
   I hope this helps a little.

Roland Halil
s635141 at uottawa.ca

-- 
John Storring   STORR96 at MEDCOR.MCGILL.CA
MD/PhD Student McGill University, Montreal CANADA
University of Ottawa Neurosci. Research Institute
Ottawa CANADA
"Everybody wants a rock to wind a piece of string around,
Everybody wants prosthetic foreheads on their real heads"- TMBG



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