Biochem_newbie

Hi there,

I am trying to see the interaction of Protein A (Myc tag)  & Protein B (Flag tag)
I transfected in 293 cells, blotted to see whole cell protein expression and came out with the expected bands.

Next step was to IP.
I pre-cleared with Protein A/G beads for 1 hour, and then transferred the supernatant into new tubes and put Myc conjugated beads overnight.
Next day I washed with NP-40 three times and then  prepped the beads with sample buffer and then boiled.

Ran a western and blotted with Flag. (IP with myc and blot with flag)
My samples were

empty vector (negative control), protein A, Protein B, and Protein A&B.
Whole cell expression was the same as before but in my IP sample lanes I see a band in lanes with Protein B (flag tag), and Protein A&B.
I know I should only see a band in the Protein A&B lane for my IP samples.

If someone could comment on this I would really appreciate it!

Thank you!

Hi everyone,

I'm currently using 293T cells to over-express my protein of interest.
I know 293T cells are used because they are easily transfectable.

My concern is that it feels like everytime I transfect, and run a Western, the expression patterns of the bands are completely different, even with same amount of DNA and protein loaded.

I am wondering, does the variation in confluency of the cells play a role in the differing protein expression patterns each time I run a Western?

Would really appreciate your advice!

Thank you