What is the purpose of input? Before I perform coip, I take some sample for input control. But input should be analyzed by PAGE on same gel with sample after coip. During this time period (when coip is performed), how do I treat my input? Should I add it to SDS sample buffer right away? Or add sample buffer to input and coip sample at the same time?
Input is a control for each solution you're adding together- in other words you can use input to determine the purity of your individual components. Basically just load the same ug you're using for the CoIP. In theory you should add SDS buffer at the same time to both your IP and input to account for degradation products/ other time dependent things but in practice you can add it to your input whenever as long as the ug stays the same for your experiment.
The total input in CoIP is a control for multiple purposes, e.g. pull-down effieciency, relative rich or enrich of your interested proteins, specificities and sensitivities of Abs & Western blotting, etc.
The amount of total input could be 5% to 10%, even 2~20% of the amount of total protein used in each pull-down, depending on how rich of your interested proteins in your samples.
In regarding the treatment of input, either of them is generally fine.
Essentially, your input matters most when your does not work. It would prove to you that you have the protein of interest in your sample to begin with. Beyond that, like Dr. Chen mentioned, it is also useful to estimate the efficiency of your co-IP.
Either of the ways you are suggesting to treat your input is fine. Personally I tend to freeze it and only add the SDS buffer when preparing to load my gel.
I am performing IP for a protein for the first time with the goal of doing a Co-IP later to study protein protein interaction. In one case after the IP when I ran the western I am having bands for the input but not IP. In another instance the band for IP is there but not as enriched as it should be. The input is giving bands though. I am using magnetic beads and and lysis buffer from a Co-IP kit. The IP protein I am looking for is a membrane protein. Any suggestions?
Hey Debanjan- Since you see a signal in the input lane, you know your protein is detectable. If its faint/absent from the IP prep, it indicates that there is the bead is not efficiently binding to the antibody. Did you incubate bead-antibody first or lysate-antibody first? I like to do lysate incubation with primary antibody O/N at 4C first, and then the next day I add beads and incubate for another 2-4 hours.
Some other troubleshooting suggestions: Try using protein A/G beads (that work better than magnetic beads), use mild detergents (NP40 based lysis buffer vs. RIPA), increase the volume of beads, etc. I'm not sure if you used denaturing/non-denaturing lysis buffer, but I don't add any SDS in my prep until the very end- when I am adding LDS prior to loading. Hope these tips help. I am relatively new to this as well, good luck!
Hi Sruti, thanks for your suggestion and tips. I used magnetic beads of Active Motif. Used the lysis buffer supplied with the kit. I will try NP40 lysis buffer for lysis and use protein A/G beads for IP. How much PMSF and protease inhibitor cocktail you add to the IP lysis buffer? What is the wash buffer that you use?
Hi, Debanjan, try PMSF at 0.5 to 1 mM. That's what most people recommend. For washing the beads I use IPP50 buffer supplemented with Pierce protease inhibitor cocktail (Thermo Fisher ref. A32965). IPP50 buffer is 10 mM Tris pH 8, 150 mM NaCl, 1% triton-x100). Keep in mind that if the protein you are IPing is around the 50 KDa and you boil everything beads, abs and your target protein at the end, you will have a big interference from the IgG used for IP, so you may need either to elute your target protein from the beads leaving the Abs attached to the beads (crosslinking the abs to the beads might help) or using a primary Ab for the WB detection made in a species different from that of your IP antibody, so that your secondary antibody will not crossreact with IgG used during IP, which of course will also be present. Good luck!
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