From Bio-Rad's user guide (see below) it is not clear to me what is the best activation time to use. It states that 1 min gives optimal results for WB followed by immunodetection, but that 5 mins activation offers the best signal-to-noise ratio. What is more important - optimal results or optimal signal-to-noise ratio?
Choose one of the following activation times based on your sample and the purpose of your experiment:
Gels used in blotting — use 1 min activation for optimal results when performing western blotting followed by immunodetection.
Good sensitivity — use 2.5 min activation when samples are abundant and when a fully optimized signal-to-noise ratio is not necessary.
Best sensitivity — use 5.0 min activation for detection of proteins that are in low concentration and for the best quantification of the maximum number of bands. Because the reaction is near completion after 5 min, this method offers an optimal signal-to-noise ratio.
Hi Gillian, we have tried 45 sec activation on the ChemiDoc MP for these gels (it's one of the default activation times on this particular imaging system), but not certain at all if this is ideal. Have you had the chance to trial the different activation times?
We were all using 5 mins but as I had a lot of gels in one experiment I cut it to 2.5 mins as a balance between the two options. It seemed fine at 2.5 mins, but if we really only nee to do 1 min, that would be more convenient.
Thank you Gillian. Do you use precast stain-free gels or do you cast your own? If you ever used precast have you ever had any issues with them?
We don't use precast but rather cast our own using the stain-free fastcat kit from Biorad.
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