Diluted ECL Reagent ?

[George W Chacko]

Has anyone tried to dilute Amersham's ECL reagent? The kind of signal
I get usually requires 3-10 secs exposure and it occurred to me that
if I could dilute the reagent in TBS-T or something I wouldn't use it
up as fast.

George

[Margaret M Lanterman]

You need to dilute your second (or First) antibody further. Also cut down your second antibody incubation (we do 30 minutes).
What you really should do is a series of dilutions of antibody to find the best for a 30 second exposure (hope that makes sense).
Actually, Amersham suggests this in their manual for ECL don't they?
Good luck
Margaret M Lanterman

[Ron Wange]

Ronald Wange
CBMB/NICHD
National Institutes of Health

[Ron Wange]

gch...@magnus.acs.ohio-state.edu (George W Chacko) writes:

:
: Has anyone tried to dilute Amersham's ECL reagent? The kind of signal

:

I have not tried to dilute ECL reagent; however, I have found
(despite Amersham's protests to the contrary) that the ECL reagent
can be reused if stored at 4 degress between uses. We routinely
use a single batch of ECL reagent for one week with good results.
Should a batch of ECL reagent go bad, one need only re-expose the
blot to fresh ECL reagent.

I suspect that your 3-10 sec exposure times are due to your primary
or secondary antibodies being to concentrated. We routinely use
Amersham's HRP-sheep-anti-mouse-IgG and HRP-goat anti-rabbit-IgG
at 1:15,000 dilution in TBST.
D
D
both Amersham's HRP-sheep anti-mouse

[fle...@guvax.acc.georgetown.edu]

I have for a long time reused the ECL reagents. Ususally a 1:5
dilution of the combined reagents in water or TE -whatever I grab at
the time. No increase in bkgd, just longer exposures. I have also done
the same with other manfacturers reagents -no problems.
good luck\

rob powell