Help.. unwanted B-tubulin in IP - (Sep/26/2005 )

Hi there,
I am having some trouble with my IP experiments. I am homogonising mouse brain tissue and preclearing my protein lysates with sepharose beads to get rid of any non-specific proteins binding with my beads. I then collect the supernatant and incubate this with my IP antibody (before adding antibody i collect some supernatant for my control). When i run these samples on a western, I get really nice clean bands of my IP products but when i probed my western with a B-tubulin antibody, i got bands in both my control and the IP samples. Thinking the B-tubulin ,may be sticky I went back and probed with NeuN a neuron specific antibody.. same thing! I would only be expecting my 2 IP products in the IP lanes not B-tubulin or NeuN. This worries me as it indicates mabye my IP isnt working (but by Ponceu stain there are fewer bands in the IP than control lanes)
Any suggestions, I need to quantify from these IPs so have to be certain my proteins are really being IP'd.

Just checking...but do you spin out your insoluble fraction before you preclear with Sepharose?

I usually let my lysates rest on ice about 30 mins then spin max spd. in microcentrifuge for 10 mins and take the supernatant for Immunoprecipitation. This removes mainly cytoskeleton and other insoluble stuff. If you aren't doing this it could be why you get "non-specific" cytoskeletal bands in your IPs.



Of course if you're already doing this, it clearly isn't the answer....
Hope this helps,

Mountainman