Western Blot with ECL

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Fri Apr 30 10:20:54 EST 2004

Alex wrote:

> Dear all,
> I now do Western blot in mouse system with ECL method. I got very high
> backgrounds when using 1st antibody, followed by 2nd antibody. So I
> did the troubleshooting. I applied the 2nd antibody only to the
> pre-blocked strips and detected. The second antibody is conjuncated
> with AP. And I get substrate from Molecular Probe. I get a very high
> background at the concentration below 1:30,000. But get less or none
> in further dilution up to 1:300,000 and 1:3,000,000. I have not tested
> for the first antibody yet. I wonder if someone out there uses such
> diluted 2nd antibody?

1:300 000 is way to high, and will almost certainly prevent detection of
your protein. If you blocked your membrane appropriately, you should not
see any background at 1:10 000 (at reasonable exposure times, of
course). Check the documentation that came with your secondary, and
select dilution and exposure times suggested. Then modify your blocking
conditions until the background is clear. Then proceed with selecting a
good concentration of primary antibody.

If using PVDF membranes, have you wetted your membrane with methanol,
then washed out the methanol with first distilled water and then
blotting buffer? PVDF is very hydrophobic and can not be wetted directly
with buffer.

What kind of protein are you using for blocking? If 5% low fat milk
powder does not work, try fish skin gelatine. Non-biological blocking
agents like polyvinylalcohol or polyvinylpyrolidone have also been

15-30 min is usually sufficient for blocking, longer blocking times may
cover your proteins and reduce the sensitivity of your assay. 

Have you included 0.1% protein and 0.05% Tween in your buffer to prevent
unspecific precipitation of antibody? Do you wash away unbound antibody
rigorously enough (3 times 15 min on a shaker, each time with at least 1
ml of buffer per square cm of blot)? Make sure that the membrane floats
freely in the buffer and does not stick to the bottom.

Is your secondary antibody ok? Check with a different batch from another

Are you using gloves and is the membrane stored protected from

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