ECL Western Blotting, need help

John Watson watson_j at bms.com
Wed Aug 6 09:56:46 EST 1997


Yankiwski wrote:

> 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.

We use Amersham's ECL reagents. I've heard reports that some of the
newer "high sensitivity" ECL kits (like Pierce's) give high backgrounds.
Proteins are immobilized on nitrocellulose rather than Immobilon, and
blocks, incubations and washes are done in TBST. Primary incubations are
done in the presence of milk, but the secondary is not. We don't use
azide (unless we are incubating the primary O/N at 4 deg C), though in
your case it seems as though it should have washed out by the time you
add the HRP. High backgrounds can result from either too much primary
(usu. lots of nonspecific bands) or too much secondary (all-over
background staining) Ab. Background problems w/ polyclonal antisera can
sometimes be overcome by affinity purification of the serum.

AJW
----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
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