Hey Kyle, nice to hear from someone from McMaster! I'm from Queen's
myself. Anyway this sounds VERY interesting because I'm finding in my
blots that I have way too much SNAP-25 and not enough B-actin. I'm
using guinea pig hippocampus, but I can't recall how the samples are
prepared. I found that I had to load only 2.5ul total protein in
order to detect SNAP-25 without oversaturation with 30 sec exposure
(from Stressgen, 1:40,000 dilution). But then when I probe for
B-actin (from Sigma, 1:1000 dilution) it have to expose the film for
10 min in order to see the bands, but they're still very light
compared to SNAP-25.
Love to hear more from you Kyle, we can move this to email if you
like.
Kyle Legate <legatek at mcmail.cis.mcmaster.ca> wrote in message news:<Pine.SOL.4.33.0301131025230.19612-100000 at mcmail.cis.mcmaster.ca>...
> In our experience this isn't the big problem. B-actin is more prevalent
> than SNAP-25 so the real problem begins at the gel loading stage. If you
> have enough SNAP-25 for the blot there will be too much B-actin. If you
> decrease the amount loaded to obtain a favourable amount of B-actin, there
> won't be enough SNAP-25. The only solution that worked for us for our
> proteins (A-actin and an anti-apoptotic protein) is to load 2 gels in
> parallel with adjusted volumes of lysate.
