Detecting BDNF with WB... - troubleshooting... (Mar/11/2010 )
Hi all, I recently have been having problems with detecting consistent, right band for BDNF which should be 14kD...my main problem is
First, I usually load 15ug to 20ug protein, but can't detect with ponceau S my protein band, but i could detect beta-actin and BNDF bands (lot of unspecific ones), should I still increase my protein amount any way? would it help?
Second, I am using 10-20% SDS-PAGE gel but having a difficult time deciding on the voltage, I would start with 100v then up to 150v because the protein run really slowly....more than two hours...but i am afraid that high voltage would distort the band...what should I do?
Third, the incubation protocol...i saw on a paper where they have really nice one band for 14KD, they use 3% BSA to block and 0.3%BSA as the solution with 1st Ab, I tried but i had huge background...
Thank you for your help!!
14 kDa is quite small, are you sure you are not running it off the gel? You are using the correct gels for this sort of thing, keep the voltage low, 150 should be fine, so long as it is not heating up. If it is heating up and distorting your bands, you can try running the gels with the tanks submerged in an ice bath or in a cold room.
Ponceau is a non-specific stain, you can't say with any certainty that any band you see on your membrane is any particular protein; it is likely to be a mixture of several of similar size.
It may be that you are transferring the protein through the membrane, you may have to try a lot of different times and transfer voltages/amperages to get the right combination. PVDF should work better for the transfer as it tends to bind the proteins a bit stronger than nitrocellulose, so it is quite common for proteins to transfer straight through nitrocellulose.
bob1 on Mar 15 2010, 07:51 PM said:
you can also use a smaller pore (0.2um or smaller) membrane to reduce blow-through.