I collected some SFEM-based conditioned medium, use a TCA-DOC precipitation protocol from here ( Article An Optimized Approach to Recover Secreted Proteins from Fibr...
) and resuspend protein in urea buffer, measure concentration and did dentauration after adding laemmli buffer like I did with normal protein. I ran the gel, did western blotting and instead of a 15 kDa band that is very specific on my control sample from cells, they appeared at around 50 kDa, but still very specific. So I did a Ponceau S staining (I know I should do it before blocking, but I forgot so I stripped the membrane and did the staining) and it showed me that at the position which I saw the band, there is a massive accumulation of protein (attached, ladder size on the left).My deduction is that for some reason the protein moved really slow in these lane so the 15kDa protein plus a bunch of other small size protein were stuck there, but it doesn't sound scientifically sound. Has anyone have this problem before?
Also has anyone seen a yellow transparent plastic-like pellet when they do their TCA precipitation? I couldn't dissolve them in acetone so I just get rid of them. Are those proteins? If so is there anyway to dissolve them?
Finally, if I am trying to detect cytokine secretion from cells, is it not wise to use SFEM?
Many thanks!
- Badges
- Science method
Similar questions and discussions