I’m attempting to co-IP binding partners from a protein. The problem is that no matter what I do, I have ”smudgy” lanes with super high background. This is a problem because I can’t see if my bands of interest appear. I am using near-infrared detection using a Li-cor odyssey scanner and the background appears in both channels, no matter which specific secondary I use.

Protocol info:

My IP protocol is pretty standard from what I can tell. I use pre-cleared GammaBind sepharose beads. Anti-myc antibody to pull down my protein of interest. Elute with a SDS sample buffer that contains bromophenol blue, DTT, B-ME, and various inhibitors. Boil at 95C for 7 minutes, spin down beads, and load the supernatant for SDS-PAGE. I transfer to a nitrocellulose membrane and do pretty standard Western procedures after that.

Troubleshooting so far:

- different beads (protein A/G Plus from Santa Cruz)

- different sample buffer containing different dye (Li-cod’s own with Orange G)

- loading beads that don’t have antibodies doesn’t help

- it’s not a heavy/light chain or antibody compatibility problem

However, NOT boiling the beads at all after adding sample buffer seems to reduce the effect a lot. Still, I really want to know what the problem stems from. We know of other labs that use near infrared detection with the same equipment and very similar protocols and have great success, and they boil their samples. Any suggestions or ideas would be greatly appreciated!

More Mohammad Amin Ghane's questions See All
Similar questions and discussions