I have different sizes of Fc fusion plasmids, I transfected with 293T cells and purified with protein A beads. After that, I run the protein gel and found that they showed the same bands. But If I run western blot using the cell lysates, I can get different bands of these Fc fusion protein.
How are you doing your purification? Is there an antibody involved? If the bands on the coomassie stain don't match the bands on the western blot (and you used the same percentage gel), chances are the bands on your stained gel are not actually your protein of interest.
You should know the expected size of your proteins so running a protein ladder next to your sample will tell you if the bands are in the correct size range. This should be the same for both your blot and your stain. If they're not, you are likely seeing an overwhelming contaminate band on your stain.
One thing that would cause this is using an antibody non-covalently attached to the beads as the antibody will elute with your protein and show up in the stain. Alternatively, there are some proteins in the lysate, such as Hsp70, which love to bind non-specifically to anything and can be difficult to remove from your purification.
Dear Shasha Alice Cheng
did you performed the transfection in 293T in a serum free media?
Can yuo show us the SDS page to undertand the MW of the repeated band?
Did you run SDS page in reducing or non-reducing conditions?
best regards
Manuele
Hi Shasha, I have the same problem with my proteins. Different size fc fusion proteins, after purification with protein A, result in the same SDS-PAGE band. Did you find any solution?
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