Firstly Thanks very much for your ideas.
I use 50 ug liver tissue, 30 ug muscle tissue and 20 ug fat tissue for western blot. I use around 10 ug or 15 ug cell lysate for western blot. Maybe you means that this protein is more accmulated in tissue.
Tissue come from mice and cells is mouse 3T3-L1 cells. so they are same species. You are right, I use different lysis buffer for tissue and cell.
I did not run them in the same gel or blot, but the gels I used were bought from the company and the same catlog number. I think both gel should be the same and the prestained marker should show the same pattern of ladders
I think I should try both in the same gel and at the same time decreasing the amount of tissue.
Do you think I need try another company's antibody?
polyfractal, on Jul 24 2009, 04:06 PM, said:
-First, the protein quantity in the tissue will be drastically higher than in the cell culture. Higher protein contents tend to run differently than low protein content. 10kDa seems a bit much of a difference, however. Perhaps try reducing the amount loaded for the tissue sample and see how it runs?
-Second, you may have different lysate conditions. For instance, varying amounts of salt or detergent will make the bands run to slightly different weights. Do you use the same lysing procedure for both tissue and cell culture?
-Are you using a stained/dyed ladder? The dyes used to color ladders affect how they run in a gel, which is why the weights are listed as "apparent weights" rather than true molecular weights. The "apparent weights" can be variable depending on the exact makeup of your acrylamide gel (acrylamide percentage, pH, glycine vs. tricine, etc)
-Post-translational modifications are not likely in this scenario. 10 kDa seems way too large for a post-translational modification.
-Perhaps you have a unique isoform in the mouse tissue not seen in your cell line? Is your cell line the same host (ie. both mice?)? If so, this seems like an unlikely possibility, at least without also seeing the 65 kDa band.
-Did you run these on the same blot or different blots? It may just have been differences in the gel composition.