The image shows 2 western blots. The first one is of protein A, and the second is for the flag tag that the protein is bound to. The lanes are as follows Ladder, Beads, SAB(unbound), and elution. THe F and E on the mid right of the image show that bands for Flag tagged protein and Endogenous protein.
I've been using Chip to pull down protein(A) with cells Flag tagged for that protein. The ChiP has been working well and I've been successfully pulling down DNA linked to protein A.
Now, I want to try and also get the protein A itself too. This should be simple since I'm already pulling down the protein+DNA, so just need to seperate the protein from DNA in elution step instead of degrading the protein. So, I've replaced the elution step(Elution buffer +proK) with Elution buffer + Flag peptide. The higher band(labelled F) is Flag tagged protein A and the lower band(labelled E) is endogenous protein A
Would appreciate any input on understanding my western on:
1.Why my SAB(unbound) still has some Flag tagged protein A?(endogenous A is normal here)
2. My eluted samples have been extracted by using very specific flag peptide for Flag tagged protein A, so why am I seeing an endogenous band.
3. The thickness of my 2nd flag western(on the right) shows a thick input band for the flag. However, the thickness of bands in elution and SAB don't add up to the one in Input so is it possible that a lot of it may still be stuck on the beads?(The beads lane reflects boiling of beads with lameli buffer and then loading that sample on SDS-Page. The actual beads may not have released all bound protein A)
Would appreciate some understanding on this. Apologies if someone finds my trajectory confusing, doing this for the first time!