Hi, I'm having some trouble running a basic immunoprecipitation experiment.
I'm trying to pull-down actin from RAW cells, but all I get is a smear throughout the entire lane with no visible or distinct bands (first image).
I ran a second experiment using the same protocol and materials but with pure actin instead of the cell homogenate and was able to pull down actin (see second image).
I'm using RAW 264.7 cell homogenates (obtained from mechanical lysis using a french press), anti-beta-actin antibodies, and protein g sepharose beads. I've been incubating the various proteins rather than a column, but again the second experiment shows that the protocol works.
Thanks for any help and advice.
I suspect protein degradation. Either by endogenous proteases or by microbial contamination. What happens when you spike your extracts with actin?
When you spike your samples with actin and you get an actin band, it means your IP is working at sample conditions, but the sample has issues. If you run the actin spiked (kept in parallel with the IP) sample on a gel and can't see the actin, you have a protease issue.
A possible contamination also could be in the IP reagents.
I mean, how do you get them off the dish? Or are they floating?
- Badges
- Science method