Western Blot for 320KD Protein - (Sep/27/2011 )
For my work i have to do a western blott for 320 KD protein. I tried 7% gel. Bought a gradient gel of 2%-16%. Run the gel overnight (4 degree). Transferred 3hr in cold room. Still not able to detect the protein.
Suggest where am failing.
Thanks
bochum on Tue Sep 27 17:01:33 2011 said:
For my work i have to do a western blott for 320 KD protein. I tried 7% gel. Bought a gradient gel of 2%-16%. Run the gel overnight (4 degree). Transferred 3hr in cold room. Still not able to detect the protein.
Suggest where am failing.
Thanks
do you have a positive control for your antibody or your western blotting technique as a whole?
Large proteins, because they are typically low abundance (the cell can't produce much of them because of the size), require that you load a lot (try titrating the amount) of protein to detect them.
did you add 0.05% sds and 10-20% methanol to the transfer buffer?
the sds will assist in getting the protein out of the gel and the methanol will strip off the sds.
Here is a useful protocol for big protein like this 320 KD and small proteins such as 30KD. See links below or the attachment.
http://www.lifetein....ll_peptide.html
http://www.nature.co...prot.2006.4.pdf
