Problems with Immunoprecipitation(Cross-linking Ig) - (Jul/05/2008 )
Hi all, I am performing immunoprecipitation on a 50kd transcription factor. I intend to use it for downstream applications(to use it for binding site selection), however first I need to confirm that the procedure works by performing IP + western blot. I am using Dynabead Protein G from Invitrogen.
I've read numerous responses on this forum about ridding the heavy and light chains of the antibody as they are co-eluted with the protein(the band of the heavy chain covers my protein), but I haven't seen any trouble-shooting threads about cross-linking the antibodies.
Anyways, I have performed the cross-linking reaction and to my dismay I was not able to detect my protein on the westernblot. I used 0.1M citrate buffer(pH 2.9) for the elution. I eluted the Protein G-Ig complex before immunoprecipitation to get rid of unlinked antibodies(the collected supernatant showed the heavy and light chain on the western blot). So I am very suspicious that my cross-linking did not work and that I eluted all the Ig before adding my protein samples.
Are there any methods that I could use to make sure that the cross-linking reactions worked? Also does anyone have experience with the use of Dynabead Protein G. The protein G is so viscous that I was afraid the suggested incubation time in their manual(up to 1 hour) for the protein sample is too low?
Thanks a lot
I've read numerous responses on this forum about ridding the heavy and light chains of the antibody as they are co-eluted with the protein(the band of the heavy chain covers my protein), but I haven't seen any trouble-shooting threads about cross-linking the antibodies.
Anyways, I have performed the cross-linking reaction and to my dismay I was not able to detect my protein on the westernblot. I used 0.1M citrate buffer(pH 2.9) for the elution. I eluted the Protein G-Ig complex before immunoprecipitation to get rid of unlinked antibodies(the collected supernatant showed the heavy and light chain on the western blot). So I am very suspicious that my cross-linking did not work and that I eluted all the Ig before adding my protein samples.
Are there any methods that I could use to make sure that the cross-linking reactions worked? Also does anyone have experience with the use of Dynabead Protein G. The protein G homogenate is so viscous that I was afraid the suggested incubation time in their manual(up to 1 hour) for the protein sample is too low?
Thanks a lot
1. You can use Trueblot to avoid detecting the native heavy chain. Does not work as great as the company claims, but better than nothing.
2. As for the method of cross-linking worked, they must have troubleshooting in the kit manual, or ask the tech rep. But off the head, I can think of crosslinking the ab, then reducing/boiling etc, and running the beads supernatant on the gel. If you see huge bands, it didn't work. if you don't see, did work! You can use some off the self crosslinked beads as control.
HTH. Let us know what worked out for you!
2. As for the method of cross-linking worked, they must have troubleshooting in the kit manual, or ask the tech rep. But off the head, I can think of crosslinking the ab, then reducing/boiling etc, and running the beads supernatant on the gel. If you see huge bands, it didn't work. if you don't see, did work! You can use some off the self crosslinked beads as control.
HTH. Let us know what worked out for you!
Yea I was kind of thinking of doing that but since crosslinking isn't 100%, I'll have to compare qualitatively with my control which would be uncross-linked beads. I guess it could work but I was afraid the band would be so thin with commasie staining that it would be difficult to tell.
Crosslinking isn't 100%. Also some antibodies won't IP if they are crosslinked.
Several things to try instead of crosslinking
1. Instead of secondary antibody, use protein A-HRP or whatever your detection system is. This should not bind denatured antibodies.
2. Don't reduce your sample, then most of the IgG will be dimers around 100 kD
3. NHS-sepharose is chemically activated and will crosslink antibodies instantly, but not orientated. Crosslinking is good, but some antibodies won't work about crosslinking.
Is it possible for you to design a specific elution, eg. using a tag and then elute with peptide. This will be cleaner.