Douglas Feinstein wrote:
>> I apologize if this has been posted previously. We get many negative
> images, or ghost bands, on our ECL exposures. Almost all the time we get
> these things with the MW standards that were used; and then with certain,
> but not all primaries. We're using PVDF, transfering in CAPS buffers, and
> using amersham's ECL reagents. I suspect we should drop primary and
> secondary amounts down a factor of 5 or 10 (now we use at 1:2000 primary;
> 1:10,000 secondary
>
yes, titration of antibodies is important for good ECL results. This is
mainly due to the higher sensitivity of ECL as compared to colorimetric
detection. We had drop the concentration of the primary by a factor of
five, that of the secondary even more (now we are using a 1:100.000 fold dilution)
Also, blocking needs to be better for ECL than for less sensitive
detections. Increase the time or change to a more efficient blocking reagent.
Frank
