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CO-IP problem: non specific prot binding to G beads - (Feb/02/2009 )

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mikew on Feb 9 2009, 08:20 PM said:


The preclear during lysis may be the problem.

I get my lysate then dilute in IP buffer. Then preclear with beads for 2 hours.
The conditions during lysis are not necessarily the same as after adding IP buffer (cell membranes, NaCl concentration etc).

Also, in your 1st post you said you transfect a myc-tagged protein into your cells. This not endogenous protein. It's possible (likely) that the overexpressed my-tagged protein binds to the beads. If this interacts with your endogenous protein of interest it will bring the interacting protein to the beads indirectly.

Did you do a control Western for anti-myc? Is the transfected myc-tagged protein also on the beads?

well, I tried doing lysis using untransfected cells on plates and preclear after spinning down cell debris. The results is the same.
i never tried the control where I check if the myc-tagged protein binds to beads as well. But the IP aMyc works, I checked on western.
I'll try to do more strigent washes next time.


rkay447 on Feb 9 2009, 11:05 PM said:

Although the method of adding the antibody to the lysate and then adding beads is popular, I really don't like it. If you are using the 9E10 myc antibody I highly recommend the agarose conjugated available from santa cruz. I published a large Co-IP using this antibody and found the prebound to be much more reliable and it was easy to determine how much of the control IgG (also available prebound to agarose beads) I needed to use. I recommend you use about 10ul of the slurry, wash at least two times in PBS and then block in freshly made and filtered 5%BSA for at least one hour but I usually went overnight. Just set up the beads the day you transfect your myc construct. Wash the beads at least twice and add your lysate. Incubate at least four hours but again, I usually went overnight. Wash beads well three times with lysis buffer and boil the beads in sample buffer.

My first trick is I use an insulin needle to remove the washes. This way I can remove all the wash, thereby getting the most efficient wash and the insulin needle is too small to suck up beads in the vacuum aspirator. I never have to worry about loosing a pellet. Another trick is to either raise the salt or detergent in the wash to eliminate non-specific binding but it may actually help to wash with a low salt buffer. Some non-specific interactions are mediated by salt-bridges that won't be broken in high salt. You may have to do much optimization to find what works best for your situation. Finally, make sure you aren't centrifuging your beads too fast. I only spin my beads at 2000rpm for a few minutes to pellet them in between washes. Faster spins can alter or destroy the bead's binding capabilities allowing more non-specific binding.

How many ug of total protein lysate are you using with how many ug antibody? Are you getting good expression of the myc-protein? How does the myc IP look? Hopefully you are checking the IP even though the co-IP isn't working yet. Do you have any positive control you could check the IP for (ie: a known interacting protein. When I IP a cyclin protein, I always check for the Cdk before looking for my (hopefully) interacting protein).

the Ip - myc looks good, I check on western the expression of protein in cell lysate and the amount of Myc-tagged protein that I bring down.
I use around 750ug of TCL per 2-5ug of Myc antibody. however I don't know how many ug of my Myc-tagged protein I have...
yes, I found a positive control, but didn't try it yet.
Thanks for advices, I 'll try it for sure.

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