how to get specific bands in co-ip - (Jun/18/2013 )

Hi everybody, I'm working on co-ip to get the interacting proteins with my target. but now the problem is no differen between the target antibody and control. I use the IgG and protein A beads as negative control. Everytime, the igG and protein A beads also can pull down many protein bands, nealy have no difference compared with target antibody. i have increse the washing times, but still no help. who knowns how can i continue with it? thanks a lot in advance! Best, Amafantuan.

-Amafantuan-

If you can provide your protocol, that will give us something to troubleshoot -at the moment, there are so many variables that could potentially be going wrong, that there is just no way to tell.

-bob1-

bob1 on Wed Jun 19 04:26:41 2013 said:

If you can provide your protocol, that will give us something to troubleshoot -at the moment, there are so many variables that could potentially be going wrong, that there is just no way to tell.

thanks for your reply! here is the protocol:

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Using rupturing buffer (100mM Tricine, pH=7.8, 1mM EDTA, 5 mM MgCl₂, 1mM DTT, protease inhibitor) to extract protein.
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Add 50 ul protein A in a 1.5ml tube, Wash protein A 3 times in 1ml pre-cooled 1×PBS(pH=7.4) .
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Centrifuge at 3000g at 4℃ for 30s to pellet beads. Discard supernatant.
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To pre-clear sample, add 1ml(1mg) protein sample to the beads, and incubate for 3h at 4℃ on a rocking platform.
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Centrifuge at 3000g at 4℃ for 30s to pellet beads; Transfer supernatant to a fresh tube.
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Add rabbit polyclonal antibody to the pre-cleared sample and incubate overnight at 4℃ on a rocking platform.
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Add 50ul protein A(have been washed 3 times in 1ml 1×PBS(Ph=7.4)) and incubate for 4h at 4℃ on a rocking platform.
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Centrifuge at 3000g at 4℃ for 30s to pellet beads. Discard supernatant.
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Wash beads 4 times in 1ml PBST (PBS adjusted to 1% Triton X-100).
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Centrifuge at 3000g at 4℃ for 30s to pellet beads. Discard supernatant.
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Wash beads 1 times in 1ml PBS only, to remove detergent.
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Resuspend beads in 100ul soft elution buffer(0.2% (w/v) SDS, 0.1% (V/V) Tween-20, 50mM Tris-HCL, Ph=8.0).Incubate for 7min at 25℃, shaking at 1000 rpm.
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Centrifuge at 3000g for 30s to pellet beads. Carefully remove supernatant, and transfer to a fresh tube.
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Repeat step 12.
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Centrifuge at 16,000g for 30s to pellet beads. Carefully remove supernatant, and pool with eluate from step 13.
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Centrifuge eluate at 16,000g for 1min, to pellet carried-over beads. Transfer supernatant (~200ul) to fresh 1.5ml tube.then SDS-page.

-Amafantuan-

OK, that looks fine - how are you detecting your co-precipitated protein? If you are just using coomassie stained gels, you probably won't be able to detect anything. It is normal to have quite a bit of non-specific stuff come across on the beads. For a co-IP it is typical to get about 10% or less of the input protein amount, given the efficinecy of the IP, whether you are precipitating all the directly targeted protein and the stoichiometry of the reaction. In other words - try detecting the protein using a specific antibody.

-bob1-

bob1 on Wed Jun 19 09:05:05 2013 said:

I want to use co-ip to find the interaction proteins. Actually it had no difference between specific antibody and IgG, so now I try to do solution digest with total co-ip proteins, then analys with Lc-MS/MS. but no idea wheather it is a practicable solution? thanks! --Amafatnuan

-Amafantuan-

My point is that you probably won't see a difference by eye as the changes are quite small and subtle in most cases. I would recommend MALDI tof-tof for protein detection.

-bob1-