I unfortunately do not have a BDNF control. This is mouse hippocampal lysate, with polyclonal rabbit bdnf antibody 1:1000 and goat anti-rabbit IRDye 800 secondary. Chameleon duo ladder and PVDF membrane. I see that BDNF is at 14kDa, dimers at 28, and pro-BDNF at 32, so what is this I'm detecting?
I think this could be the proBDNF signal. While the BDNF should appear below 15 KDa. You can try thicker gel probably 15% gel to catch the signal or the protein would run down.
an you give more details? is BNDF charged? acidic or alkaline? the treatment matters in these cases
The high molecular weight bands may be the glycosylated BDNF or other post translational modifications.
I concur that you need to run a 15% gel to tag mature BDNF, and that this is likely ProBDNF, but I'd need more info to be certain...
Did you do a workup on this antibody with a serial dilution? (Can you tell us the manufacturer and catalog number?) How much protein did you load and what was the incubation duration (i.e. RT for 2 hours or O/N at 4C?). Is the protein fully denatured and in BME?
More info, using 10 well 12% bis-tris but am fairly sure im not running the protein out of the gel as my die marker is not yet run out. Samples were lysed in RIPA + PPI, and quantified using BCA. Loaded ~100ug of protein per well, incubated in primary overnight at 4C. No BME, reducing agent was NuPage reducing agent (DTT). I have since found several other papers that either report BDNF detection at 18kDa or state that BDNF has a movement weight of ~18kDa, as well as several antibodies that show an 18kDa band assumed to be a mature BDNF isomer.
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