In article <5gnb8r$4fm at news.doit.wisc.edu>,
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote:
> Hello all,
>> our lab is finally moving from I-125 to ECL-based Westerns.
> While I am sure every kit works, I am wondering what you guys
> would recommend to use most. Right now we can think of a reagents from
> Boehringer, Amersham, Pierce and NEN. Of course, other things
> being equal, the cheaper the better.
>> Everyone who actually compared different "kits", please reply.
>> TIA,
>> Dima
Hi
I have made some fine looking western blots with Amershams ECL system,
and one can use much less of the reagents than they recommend, as has
already been discussed here several times. Still the Peroxidase/Luminol
systems have a rather annoying drawback. The signal is very intense but
short lived. That means that I wouldn't trust any quantitations made with
ECL and it's also a waste of X-ray film most of the time, while finding
the right exposure time.
We use the Alkaline Phospatase substrate CSPD for southerns and like it
a lot. The emission is much more stable than with ECL and much more
quantitative. There are some Western kits around based on CSPD or its
more intensely luminous brother CDP-star, but the ones I have seen
(Tropix) are just to expensive. I tried a protein dot-blot using and CSPD
from Boehringer for detection (Boehringer sells both substrates at
reasonable prices) but I was not happy with the sensitivity. So now I
would like to add some questions:
Does someone have a good protocol for Westerns with CSPD or CDP-star?
Are there any affordable kits around?
Any information, no matter how trivial is appreciated !!
Zophonias
_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie Tel: (41-1)-257-54-75
Universitat Zurich-Irchel Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
_____________________________________________________________________
