ECL Western Blotting, need help

[Johan Lennartsson]

Hi

I've found that when I block the Immobilon membrane with only 0.1-0.2%
Tween-20 in PBS overnight at +4oC or 2 hours at room temperature and do all
washing in 0.5% Tween-20, it works very well. When I stripp my membranes I
use 55oC for 30 min.
Another thing that I found to be important is to use large volumes for
washing and not wash to quickly (200-300ml PBS-T for 10min, 3-4 times).

Good Luck
Johan

Yankiwski <yankiwski at aol.com> skrev i inlägg
<19970731001900.UAA22820 at ladder02.news.aol.com>...
> 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
>