I have done western blot to find GDNF protein band but I am not sure the bands which I got in western are real GDNF band or they are not specific bands. I want to explain experiment condition.
I load 40ug of protein in 12% polyacrylamide gel. for transfering I Used PVDV membrane and voltage of 200 mA for 1 hour. I dilute rabbit polyclonal anti GDNF antibody(abcam) with ratio of 1-1000 in 5% skim milk in TBST and incubate it for overnight.
Finally, I got the band but it appear in size of more than 55 kDa, I tried on the other membrane and got the same result now, I am not sure it is GDNF or not? Because the size of GDNF in antibody catalog is mentioned 24 kDa and in some papers mentioned 33-45 kDa.
I would appreciate if anybody can help me in this matter.
Hi, I can try and help with this one. We have tried many different anti-GDNF antibodies (not Abcam though), and sometimes a different lot of the same antibody will give a different answer. The R&D goat (AF212-NA) is the only one that gives us the 15kD Mwt mature form that we want, but this is a difficult antibody to work with, lots of background etc. It sounds like you are getting the a pro form of this growth factor. Do you want to send a detailed protocol and pic of what you are getting, and I can try and help you troubleshoot?
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