ECL Western Blotting, need help

Martin Offterdinger a8803349 at unet.univie.ac.at
Thu Jul 31 03:36:41 EST 1997


On 31 Jul 1997 00:19:25 GMT, yankiwski at aol.com (Yankiwski) wrote:

>Hi,
>
>I've been trying to detect a protein transferred to Immobilon membranes,
>and have had poor results.  On one attempt I got lots of bands with high
>background levels and nonspecific binding.  Stripping the membrane and
>probing again with lower Ab dilutions resulted in a blank film.  I've
>tried a variety of Ab dilutions and incubation times, without better
>results.  Is there a subtlety in the technique that I'm missing?  The ECL
>detection reagents themselves are working properly, but perhaps my
>stripping conditions (30 min. @ 65 C) were too harsh?  The only other
>reagents I use are 5% non-fat dry milk in TBS w/ NaN3 to block, and
>TBS/0.1% Tween-20 for washes.  (The Abs themselves are diluted in non-fat
>dry milk in TBS).  Any suggestions on improving my results and on the ECL
>technique in general would be appreciated, as I am new to this method. 
>
>Thanks,
>Victor
Dear Victor!

1. Are you shure that your antibody is pure/specific enough?
Because if you are using a bad antibody, probably every attempt to
improve your result will fail. In my experience there are antibodies
which just do not work. If this should be the case(many unspecific
bands, background) it will save you a lot of time to stop using this
antibody for Westerns.In my experience it does not make any sense to
try anything, if your antibody is not good enough. If you are using a
polyclonal, you could affinity purify it.

2. Stripping of westerns does in my experience only work if the
expression of your protein of interest is quite high as you tend to
remove proteins from the membrane.

3. try drying the PVDF membrane completely as this fixes the proteins
to the membrane (You have to rewet with MeOH afterwards!!)



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