I am using anti-fibronectin antibodies in western blotting experiments, and in one breast cancer cell line, I found ~230kDa bands, but in other cell line of this cancer, I found bands of ~35kDa ... How is it possible? I read that fibronectin has 30kDa n-terminal fragments... is that the reason of my finds? ( thanks and sorry for the bad inglish)
Oi Alexandre!
I see 3 possible explanations to this situation:
1- Sometimes is complicated to detect expression of a protein across cell lines ran in the same gel and developed at the same time (Is that your case?). The levels of expression for this particular cell line may be too low compared to the other ones and you would need much more time of exposure of the film when developing it. You may need a specific time of exposure for each cell line depending on the expression levels. However, this impede the direct comparation between bands from different cell lines. Have you tried different exposure times or amount of loaded protein?
2- The 35KDa band could be a non-specific band. Do you see it in the other cell lines although very faint? As you said, it could be the n-terminal fragment of Fibronectin and it could become detectable if your protein is suffering degradation. To rule this out, check if the epitope the antibody detects is located in the n-terminal part.
3- Another possibility is that this particular cell line may display a mutated form of fibronectin that does not allow for the epitope recognition. It happened to me once.
Espero ter ajudado!
Boa sorte!
Hi Alexandre,
Although, all the 3 points suggested by @priscila marcondes are possible explanations for your case. Considering that you have loaded equal total protein amount for both cell lines, I think relative protein expression level and/or degradation could be the main reason.
I hope it will help.
Hi Alexandre!!
Depending on the strains you are using, they may express the protein differently. It would be nice to test different protein concentrations, to verify that your 35KDa band is not nonspecific, as this happens a lot in this technique. In addition, I recommend testing different exposure times for your membrane on the photo-documenter!
Hope this helps!!
- Badges
- Science topic