I want check the protein level of BMP2 (Bone morphogenetic protein 2). I see someone use cell lysiate to do western blot while others use cell culture supernantant to do ELISA for BMP2.
I know BMP2 is a secreted protein. Is it correct to check such kind of protein using cell lysiate (although someone did so)?
If you have done this BMP2 or any other secreted protein check, can share the experience with me?
Thanks!
Yes, you can, but you will need to incubate the cells at the presence of brefeldin A before preparing the lysate. https://media.cellsignal.com/pdf/9972.pdf
if it is secreted, why do you want to see what is still within the cells? you should collect media (serum free) and precipitate proteins using ethanol or acetone. the you can resuspend protein pellets in loading buffer and run westerns.
Hi Derek,
The thing is, for this protein, I saw some people use cell lysiate but others use ELISA. Both kind of method can be found in publications. So the question is asked by myself. I am still in the designing stage. I don't know whether use cell lysiate is ok. If it's not ok then I may change to study another gene cause this is only one of the genes from microarray that seem interesting.
As you mentioned, I'm not sure for my cells, if I culture with serum free medium, they can grow or not. If cannot grow, then what should do? Another thing is I don't know whether the protein in the supernatant is enough to do western blot. You even done this before? How about the concentration of your protein in the supernatant?
Thanks!
- Badges
- Science method