Can my sample be going to the buffer instead of the membrane?? - 17kDa protein not showing in WB, transfer is fine though. (Sep/15/2009 )

I am completely puzzled by this. I'm doing a western blot on a 17kDa protein after affinity purification by HPLC. My transfer is fine, the ladder transfers nicely, and the commasie stain on the gel is negative. I thought I was not loading enough protein and did a titration curve with the standard protein I bought. Well, the same thing happened! 30ug of std protein were not visible. The gel was empty so transfer was fine and my ladder was showing up in the blot. I even incubated ovrn with the 1ry ab @ 4C. Could my protein be floating in the buffer and never stick to the membrane?

I use Nitrocellulose (pre-wet in transfer buffer)
20V for 5o to 60min

The whole film is clear after developed!

Put a membrane on both sides of the gel, and check them both -- if the protein is positively charged under the conditions of your blot, it'll go "backwards". Also, could a 17 kDa protein blow through your nitrocellulose? Perhaps you should try a membrane with a tighter matrix, like nylon or PVDF.

I thought so too, but I read someone here is using nitrocellulose on a 15kDa protein, so I don't know what to think anymore. I'll do a ponceau stain on the blot to see if my Abs are not working anymore which I doubt. I could try the double membrane though.

HomeBrew on Sep 15 2009, 01:56 PM said:

However, they should be neutral with SDS.

medchemgirl on Sep 15 2009, 02:05 PM said:

SDS in PAGE causes the proteins to denature, and the SDS then binds to the proteins, giving them a uniform negative charge per unit of mass. Since all the proteins are now uniformly negatively charged, SDS-PAGE seperates the proteins based on size and is not influenced by the diferential charges found on native proteins. The negatively charged proteins migrate to the positive pole.

You're also relying on this negative charge to draw your proteins out of the gel and towards the membrane in a western blot. If, however, the protein is positively charged for some reason under the conditions of western transfer (a not unheard of occurrence), the protein will be drawn out of the gel in the opposite direction.

You can sometimes overcome this by adding SDS to your transfer buffer, or by placing a membrane on the other side of the gel.

Adding SDS, as homebrew suggested, may be better, as the protein will be on the same membrane as your markers.

for a small protein i would not add sds to the buffer, that is done to facilitate transfer of large proteins. the protein is exiting the gel just fine. i think that the protein is blowing through the membrane (test this by putting a second membrane behind the first)

what porosity nitrocellulose are you using? we use 0.2um pore nitrocellulose for small proteins.