j.abbott at dundee.ac.uk
Wed Sep 10 14:37:33 EST 1997
curtis alexander wrote:
> Can anyone suggest a reliable, sensitive and relatively quick non
> radioactive method of detection? Any help will be appriciated.
We have tried a couple of different kits, generally fairly succesfully. For
Westerns, both ECL kits from Amersham and a kit from NEB seem successful, the
NEB kit produced fewer artifacts and less background than ECL, but Amersham
have since produced a new version of the kit which is meant to be better.
For Southerns and colony blots, we have used ECL again, although this hasn't
been as good for low target concentrations. In one case we also tried
Amershams Gene Imager kit for use in low target applications but without
By far the best results have come using DIG labelling from Boehringer
Mannheim, although using the kit as directed did not produce particularly
impressive results. Following the protocol of Blum et al (1993) (see below
for reference) produced startlingly good results. The blot used with
Amershams Gene Imager was stripped, and reprobed with a DIG labelled
riboprobe and this protocol and produced bands we had not been able to see
using 32P - very impressive stuff, and completely safe.
The reference for the protocol is below:-
Authors - Blum G.E., Meier, M., Frank, J., Muller, G.A.
Year - 1993
Title - Reduction of Background Problems in Nonradioactive Northern and
Southern Blot Analysys Enables Higher Sensitivity Then 32P-Based
Journal - Anal. Biochem
Volume - 210
Pages - 235-244
Hope this is of some use.
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