Hi there,

So you are studying a protein interacting with actin. If this protein is containing Ig or Ig-like domains (which is often occuring in actin partners), it might have an affinity for protein G which also interacts with Ig domains. So your complex of interest might interact with beads even without any added antibody.

Had very hard time with actin co-IP (same buffers), half of the times I had non specific binding. The best solution for me was to tag my target protein (FLAG/HA etc) and elute it with a peptide.

one of the possibility is that in your buffer condition, actin form filament which are seasely trapped within the beads...so perform a cleraing of you wce and or add cytochalasin (an actin depolymeriser) in you extract buffer...and work carefully, either spin the agarose beads no more than 3-4Krpm or use magnetic beads..

Hi Aaron,

There are a couple of things you could do some of which have been mentioned already.

1. Increase the buffer stringency. Usually higher salt conc. helps

2. Pre-clear lysates with the beads

3. Do not use excessive quantities of beads, this provides free surfaces for nonspecific binding

4. If your IP incubation is being run @4oC/overnight, you might want to reduce this to about 2-4h.

5. Ideally use magnetic beads. If not spin at very low speeds (e.g. 2000g/2mins). This is particulary important during the post-bind spin to collect the unbound supernatant.

hi Aaron, I had similar problems with protein G-agarose. I used similar buffer for co-ip... At the end it helped me to switch from G-agarose to A-agarose...

Dear Aaron,

We use the same buffer for IP and do not have actin as a non-specific contaminant.

See Proteomic Analyses Uncover a New Function and Mode of Action... (Figure 3 and 5) and Article Quantitative Proteomics Illuminates a Functional Interaction...

Nether yuor beads nor the Abs are the problem. You should simply pre-clear lysates by spinning at 15k for 10 min at 4C and then incubating with empty beads for 30 min.

Best of luck!

Metello