I am getting very faint bands and cannot seem to increase the signal.
I have tried increasing the amount of protein per well, increasing the primary antibody concentration, 0.45 and 0.2 um nitrocellulose membranes, different transfer settings, different incubation times and different ECL detection kits.
I would like to get in touch with someone who has had success using proBDNF (5H8) or any tips or tricks to detect BDNF by western blot.
This antibody sucks. It doesn't work even at 1:100 dilution. I like their H-117 the best for Western blot but it has been discontinued. I am still searching for a good replacement.
Has anybody observed 20-22 kdA BDNF band along with 14kda mBDNF, 28 kDA truncated BDNF and 32 kDA Pro-BDNF? What is this 20-22 kDA BDNF band?
It may be a bit late, but I also had a disappointment with this proBDNF Ab and I'm working with rat brain. I get multiple bands at 1:600 dilution (using HRP ECL) but none of them have the correct size. I add some images.
Now, I am using an antibody that has been discontinued by Santa Cruz Biotechnology but it works well for me: sc-33904 at 1:1000 dilution.
I have tried the DSHB antibody for mature BDNF but I also got multiple bands.
Although the problem didn't get solved in my case so far. But I read transfer conditions can help. Do try 60 min wet transfer at 100V. The samples should be electrophoresed using 15%gel. My antibody N20 santa cruz could detect 15ng of mBDNF peptide loaded as control on the gel at 14kda , however didn't detect any mBDNF at 14kda for hippocampal lysate and N2a cell lysates loaded. Best of luck to you!
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