On Apr 29, 1:10pm, Isla Browning wrote:
> Subject: virus detection by chemiluminescence
>> I would be grateful to hear from anyone who has tried Western Blotting
> using a Chemiluminescence Reagent (eg DuPont RENAISSANCE) for
> detection of viruses particularly those of potato. I am interested in the
> sensitivity found compared with other antibody based techniques and
> any practical problems.
I can't address the potato aspect, but I ahve used chemiluminescence with
several animal viruses. In terms of sensitivity, I have found the light to be
quite sensitive. Existing WB protocols need to be modified.
For example using a similar anitgen/antibody system, when moving from I-125
protein A detection to chromogenic substrates, we had to dilute the primary
an additional 5 fold for comparable sensitivity. When we moved to chemi, we
diluted an additional 4 fold after again. Another pecularity I have found is
that the manufacturer's suggested dilutions of secondaries are never enough.
I recall that Amersham recommended 1:1000, but we dilute 1:20000 - 1:50000
for best results.
The trick to tell which is too much (which is usually the problem with these
more sensitive methods) is to discern the nature of the background. If too
many bands appear, dilute your primary; if the overall background is too
dark, dilute your secondary.