4 replies to this topic

[bochum]

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

[casandra]

bochum, on 27 September 2011 - 09:01 AM, 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?

[bob1]

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.

[mdfenko]

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.

[LifeTein Peptide]

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

Attached Files

•  nprot.2006.4.pdf   999.21K   525 downloads