I am currently trying to isolate a protein that is above 800kDa, a fairly large protein. I have done the SDS PAGE and membrane transfer a few times. After the membrane transfer, each time I stain the membrane (Ponceau & Coomassie) I get no bands. This meant that transfer time is not sufficient (which is one of the problems I am working on).

But the main problem is that when I've stained the gels (Coomassie) I only sometimes see bands on the gel. When I did a 6% gel, I saw a few bands at the top of the gels, but only in a couple of the lanes. When I did a 4% gel, I didn't see any bands on the gel at all, but there was staining at the bottom of the wells (I also stained the stacking gel this time).

Each time I ran the gel at 200V for 1.5 to 2 hours.

Any help of suggestions would be very much appreciated.

Thanks,

Joshua Dias

You need to check and see if a protein that large will even run into a SDS gel. I routinely run 4-20% gradient gels to detect apoB-100 (512 kD) and it barely enters the gel usually running just below the well even after an hour or so. Maybe if you run a 4% gel and continue to run it for 1 -2 hrs, or at least until you've run the top marker as far as it will go, you will detect your band.

Another option for you is to run an agarose gel-large protein format.
Best of luck
let everyone know what you find out.

Rb

I will try again with the SDS gel and run it for longer. But I have a question in regards to your suggestion of the agarose gel.

I was thinking about that as well, but I didnt think it was possible to do immunoblotting with an agarose gel. What Im doing is isolating an 800kDa protein from muscle tissue. I have custom made a few antibodies whose antigens are different amino acid sequences of the protein. What I am trying to do is test these antibodies to make sure they first of all, bind to the protein, and secondly bind to the right protein.

Any further suggestions would be much appreciated.

Joshua Dias

R Bishop said:

You need to check and see if a protein that large will even run into a SDS gel. I routinely run 4-20% gradient gels to detect apoB-100 (512 kD) and it barely enters the gel usually running just below the well even after an hour or so. Maybe if you run a 4% gel and continue to run it for 1 -2 hrs, or at least until you've run the top marker as far as it will go, you will detect your band. Another option for you is to run an agarose gel-large protein format. Best of luck let everyone know what you find out. Rb