I am using a Millipore Catch and Release IP kit for Co-immunoprecipitation. As per the instructions of the kit, I am adding 500ug of my tissue supernatant to the column. While the flow through does contain a similar quantity of protein to what was loaded, the elutes for WB have only 25% of the concentration and thus require a very high loading volume. This poses a problem in the SDS-PAGE using a mini-gel system. Is this common in Co-IP? Can I increase my protein concentration in the elute in any way so that I have more accurate loading in SDS PAGE?
IP is a fishing technique directed against a specific protein which relies on the efficiency of the antibody used and the abundance of the target protein. When you consider Co-IP the chances for getting a protein to be caught which is again bound to another cross reacting protein is very slim and it happens like that way.
Try loading more samples in the column or load concentrated samples and play with primary and secondary antibodies concentration and duration of incubation while developing WB.
Try direct probing for the target protein in the loading sample and also in the flow through and carefully compare the data by normalizing against a suitable loading control to have an idea of binding efficiency.
Thank you sir. How can i concentrate my samples more? I am preparing a 5% homogenate and in the total recommended volume for the columns provided in the kit is 500ul. My sample constitutes almost 30-40% of this volume and the provided antibody ligand is only 10ul of this volume as given in the kit instructions. Can u kindly suggest a way for me to either increase the samples before IP or to concentrate the sample in the elute so as to lweor the volume which i am unable to load in the gel. Thanks
Another suggestion is to increase the concentration of proteins in your homogenate by either using more cells or tissue or use less buffer for the homogenization. That should increase the concentration of the protein targeted by the primary antibody.
If the only need for increase in the protein concentration is to increase the signal via WB analysis (e.g. because you have problems with detection of the target protein), then the simplest way is to use TCA (tri-chlor-acetic acid) precipitation. This will increase concentration of the eluted material and, subsequently, reduce the volume of the sample to be loaded onto the gel. The protocol is very simple:
Do IP in a usual way, elute the material from the beads and add TCA to the eluate (10% final concentration, v/v). Keep the sample on ice for 30 min then centrifuge at maximum (13,000 rpm) speed at +4oC. Take out the super and do not touch the pellet (if visible, but remember pellet may not be visible in many cases, so do not aspirate, use pipette to remove the super). Wash the sample once with ice-cold acetone, centrifuge again and remove the super with a pipette, air-dry your sample in a hood for 10-15 min. Resuspend the pellet in 1X Protein sample buffer. If the color of the BpB (or other pH sensitive dye in the PSB) changes to yellow, add 1-2 ul of 1.5M Tris pH 8.8 (to neutralize the traces of TCA). Heat the sample at 95oC for 5 min and run it on the gel.
Good luck.
Thanks Olego for a very detailed and easy to do method...just one more question ...my elute volume is ~80ul...which means, i would add abt 8ul of TCA to it...how much should i add the prtein sample buffer (by which i assume u mean the loading buffer)...kindly clarify so i can apply this protocol immediately
I suggest using 10x Laemmli Sample Buffer from Ecotech Biotechnology. You do not need to dilute your protein too much if you use this buffer.
https://ecotechbiotech.com/laemmli-en
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