SUMMARY: Chemiluminescent detection of Western Blots: Preferred Method

William R. Morgan wmorgan at ACS.WOOSTER.EDU
Fri Jul 30 13:43:40 EST 1993


Summary of Recommended Non-isotopic Detection System for Western Blotting:

Earlier I asked:
>Any recommendations on the preferred method for nonisotopic detection with
>Western blots?
>
In general, people seemed to have had success with the ECL system, which
uses HRP-antibody conjugates, although one person clearly preferred using
alkaline phosphatase-antibody conjugates.  

A summary of the responses follow:

dmartin at crc.ac.uk (David Martin x3175)writes...

>We have had excellent results using Amershams ECL with a variety of western
>blot types (Ab-HRP, Ab-Biotin:avidin-HRP, ligand-biotin:avidin-hrp)

>Just wash in TBS or similar (ie no gelatin) prior to adding the ECL reagents
>after your usual probing method and you should get good results.

echeverri at sci.wfeb.edu (Chris E.) says...

I would like to second this: I've been using Amersham's ECL detection 
system for years, and it is great... 

Fred Garbrect <fgarbrec at post.its.mcw.edu> writes...

>You will find that the ECL based system provides much superior sensitivity, 
>but may also be plagued by background problems.  Depends (as far as I can 
>tell) on the specific antibodies that you are using.  I would suggest using 
>the HRP conjugate initially to see if it meets your requirements without 
>too much background.

However, D.R.Micklem (drm21 at mbuk.bio.cam.ac.uk) writes...

>I've tried both Amershams ECL kit for ECL using HRP and the Western Light
>Kit from Tropix using AP.  I found the latter far better, with lower
>background but I've only tried the kits on blots for one antigen/primary
>antibody combination so its hardly exhaustive.  Another advantage of the
>Tropix kit is that I think you can get all the components separately from
>chemical suppliers to save money, since it says what they all are.  They do
>two AP substrates: I used CSPD.

Thank you all for your responses to my first posted question,
Bill Morgan


P.S.  The following recent comments, while not directly in response to my
question, also bear on the ECL system with Western blots, so I thought I'd
tack them on to the end of this summary:

dotzlaw at ccu.umanitoba.ca (Helmut Dotzlaw) writes...

In article <238v67$98k at samba.oit.unc.edu>, plc at med.unc.edu (Philip L. Carl)
wrote:

>      Recently we have switched to enhanced chemoluminescense
> to detect proteins on Western blots and have been generally
> pleased with the increased sensitivity.  However our blots
> often appear "blotchy" with irregularly shaped dark regions.
> We believe this is due to small amounts of gel that adhere
> tenaciously to the blots after semi-dry transfer, although
> we haven't ruled out that there may be other causes.  I was
> thinking of putting a little glycerol into the transfer
> buffers to make the membrane a little slippery.  The problem
> seems to be worse with nitrocellulose compared to PVDF
> filters, but we see it with both.
> 
I find that this happens with Amersham's ECL kit if the secondary antibody
is not washed long enough and there is still some reagent left on the blot
when you wrap it to expose to film.  The five washes after the secondary
are very important to dilute it out - any of the enzyme left in solution
will cause background problems.  I also take the blot and dry it with
KimWipes before putting it in the wrap, this makes a big difference as
well.

...and continues in a subsequent message...

Tried something different today and was impressed by how well it worked, so
thought to share.  A colleague suggested a variation on Amersham's ECL
detection to minimize the background on long exposures, and also to use
less reagent per blot. 

Following the washes after the secondary Ab, take the blot out of TBST and
pat it "damp" (no puddles) with some tissue, then place it protein-side
down on a piece of plastic wrap.  Layer the reagent mix (2 ml for a 4x8 cm
minigel) on top of the blot and wait 1 minute.  Pat dry again, wrap, and
expose to film.

The result - a 1 hour exposure with absolute zero background, a "first" for
me!

BTW, I used Nitroplus 2000 nitrocellulose membrane, not a "plug", just what
I have.





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