I need help with my IP protocol. I'm trying to elute with glycine 0.1 M at pH 3. I have used different antibodies immobilized (crosslinked) to magnetic beads. I'm using 10 volumes of glycine buffer to elute and then,15 min at RT with gentle rotation. I transfer the supernatant to fresh tubes with 10-20 µl of Tris-HCl 1 M, pH 7.5.
When i perform my blots, nothing!?
PS: I also elute with SDS PAGE buffer (with 2-mercapto) and this elutes the Ig chains
After elution in Glycine, you can boil the left over beads in SDS buffer to check on the gel whether your antigen is still bound after the glycine elution. Some antigen-antibody associations are not released with glycine.
Two potential problems immediately come to mind:
1) You don't have your target protein bound. Do you see your protein after you elute with SDS buffer?
2) The eluted protein is too dilute in your elution buffer. Do you load the entire volume of eluate? You may try a TCA precipitation to concentrate your eluate.
Have you checked if you are eluting any protein from your IP? Just running the samples on a gel and silver staining?
After elution in Glycine, you can boil the left over beads in SDS buffer to check on the gel whether your antigen is still bound after the glycine elution. Some antigen-antibody associations are not released with glycine.
I forgot to mention that my problem is my target protein has the same weight that Ig's, for this we have to crosslink.
We have tried to boil the beads with SDS buffer after elution with glycine but the Ig's were eluted and is impossible to know if my protein is there.
We checked the elution in gel with Comassie and we haven't see anything, may be as you suggest i have to try to precipitate (could you help me with this assay? I have never performed it)
I suspected that was the reason you were crosslinking. Did you know there is a secondary antibody that doesn't detect SDS-denatured IgG. This is an example of it, but you can get for other species too - http://www.abcam.com/veriblot-for-ip-secondary-antibody-hrp-ab131366.html
I didn't know about it. It seems a good option for my problem. Thanks!!
Amanda's solution will probably be the best for you. Let me know if you want to try the tca precipitation. I can give you the protocol.
Alternatively, you can couple your detection antibody to biotin and do the blot with streptavidin-HRP.
My protein is 24kD and I face exactly the same problem. however, sometimes if i overexpress an HA tagged version of the protein and perform HA-bead (Roche) IP and elute with following protocol it helps to reduce background and discern my band: you can try this. Good luck: Glycine Buffer Elution:
In this procedure the complex can be eluted from the beads by acidification. A glycine buffer containing 0.1-0.2 M Glycine pH 2.0-3.0 can be used. The low pH of glycine buffer helps to weaken the interaction between the antibody and the beads. This method is advantageous as beads can be reused after removal of the glycine buffer. However the eluted sample should be immediately neutralized with Tris, pH 8.0-8.5. After standard IP, follow these steps for glycine elution:
1. Elute the beads (50 µl) 3 X with 150 µl 0.2 M glycine pH 2.6 (1:1) by incubating the sample for 10 minutes with frequent agitation before gentle centrifugation.
2. Pool the eluate and neutralize by adding equal volume of Tris pH 8.0.
3. Repeat these steps 1-2 times and collect all the eluate.
4. Neutralize the beads by washing 2 X with 150 µl lysis buffer (without detergent) and pool with eluate.
5. Run the samples on a western blot to check the precipitation of proteins.
I have a similar problem for IP.. Can you help me with a protocol for TCA precipitation?Thanks a lot!
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