ECL Western Blotting, need help

Thomas Urbig at biokemi.su.se
Wed Aug 13 08:54:09 EST 1997


In article <19970731001900.UAA22820 at ladder02.news.aol.com>,
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

In addition to the suggestions you already got: blocking PVDF is harder
than nitrocellulose. I would suggest to use nitrocellulose for a better
signal to noise ratio. Blocking with BSA (pentax fraction 5) gave better
results in all cases I had before (blocking buffer 5% (w/v) BSA in 1xTBS
and 0.2 % (v/v) Tween 20 for 15-30 min). I also incubate the first ab in
blocking solution, the 2nd in TBS only. I use the 2nd ab only once, it´s
to cheap to screw up important bots although I have used it for routine
applications up to 8 times! All blots I have seen so far on Immobilon had
a high background.
Make shure your protein is really on the membrane. When blotting put a 2nd
membrane bhind the first one to check that the protein isn´t blottet
through the first membrane. Silver stain the gel after blotting to check
how much protein remains in the gel matrix. To establish blotting
conditions the use of rainbow coloured prestained markers makes life
easier. Check protein transfer with Fast Green stain (works only with NC,
not with PVDF).
At last, try another detection method. ECL is very sensitive, the use of
alkaline phospahatase in a detection system with NBT and BCIP gives high
sensitivity (OK, not as high as ECL but in most cases high enough) and
easy application and control of the detection reaction.
At last, adjust the stringency of your washes to your antibodies, play
around with the AB dilutions. As a standard protocol I use 2 washes of 2
min after the first ab and 5-7 washes for 5 min after the 2nd ab. Adjust
stringency of your wash buffers (more salt, more Tween-20, maybe add
Triton X-100 in addition to Tween) to suit the application. Don´t wash for
extended times, better change buffers more often (I discard the buffer of
the first wash usually after 1-2 mins.). Incubation of the 1rst ab o/n
might help to get a better signal but I have seen bots with monoclonals
which just produced very faint results independent of all the conditions
we tried.

Hope that helps

Thomas

-- 
Thomas Urbig
Urbig at biokemi.su.se



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