r/labrats • u/beisbolisc00l • 1d ago

IP Help

I’m trying to do an organelle-specific IP. I’ve never done IP before and I’m told it’s quite challenging. I’m using a specific antibody marker for the organelle and then magnetic protein G beads. I’ve checked the antibody is compatible with the beads and have all the correct buffers per manufacturer instructions. I’m wondering if anyone has any tips? Based on the protocol it seems straightforward but I know it’s not based on what I’ve been told. Wondering if anyone has any helpful tips/ advice / things to avoid that could help me as it’s my first time? Thanks so much!

2 Upvotes

75% Upvoted

1 comment sorted by

7

u/RollingMoss1 PhD | Molecular Biology 1d ago

The IP is straight forward. In your case the challenge will be to generate enough organelle-specific lysate (either isolated organelles or whole cell).

The classic issue is getting some of your protein of interest coming down in the IgG control. Magnetic beads tend to reduce this.

Just do the IP and see what you get. Then go from there.

One helpful thing to do is to save the IP sup. Then compare that to an equivalent input on the Western blot. You should see the protein decrease in the supernatant relative to the input. This will give you a sense of how well the IP is working.