advice needed for IP (revised) - (Mar/19/2007 )
(Due to complaints of the length of my previous post, I summarized it as follows:)
Three questions about IP:
1) method of mixing during the overnight incubation? Taping the 1.5ml eppendorf tubes containing the lysate and antibody on a platform rotator or using a vortexer at a low setting?
2) order and length of incubation? choice 1: incubate the antibody and lysate for 1 hour, and then add protein A-agarose IP reagent for overnight incubation. choice 2: incubate the antibody and lysate overnight, then add protein A-agarose IP reagent for 4-hour incubation. (all at 4 degree)
3) time of boiling after IP? 10 min or 3 min?
If anyone is interested to know the details of my experiment, please see the following text:
I recently started to establish the conditions of IP for detecting the protein of my interest. Here is what I did:
- I found two antibodies targeting this antibody (from different species), and confirmed with WB that they do recognize this protein.
- I then used a cell lysate that has been confirmed to have this protein, incubated the lysate (100ug) with one of the antibodies (1.5ug) overnight at 4 degree, the next morning, add protein A-agarose IP reagent (20ul) from Santa Cruz (which should recognize IgG from the species the antibody is raised), incubate for another 4 hours at 4 degree.
- after the incubation, I centrifuged the mixture at 1000Xg for 5' at 4 degree, then washed with 1ml PBS buffer (cold) four times, each time, centrifuged as above. after last wash, added 25ul RIPA buffer and 2ul 20% SDS, boiled for 10'. then short spin and used ~13ul supernatant for WB, in which the primary antibody is the one from the other species.
With 20ug of the above lysate and the second antibody, I was able to see a lot of the protein of my interest by WB. The IP I described above used 5-times lysate as I used for direct WB. After IP, I loaded ~half of the supernatant. Theoretically I should see a fat band after WB of the IP samples. However, as other things in life that don't go theoretically, I saw a much fainter band. I also analyzed the supernatant after the first centrifugation before washing, there are still at least half that protein left behind.
I have been really frustrated by this issue recently. Any advice would be really appreciated! Thanks a lot!
I recently started to establish the conditions of IP for detecting the protein of my interest. Here is what I did:
- I found two antibodies targeting this antibody (from different species), and confirmed with WB that they do recognize this protein.
- I then used a cell lysate that has been confirmed to have this protein, incubated the lysate (100ug) with one of the antibodies (1.5ug) overnight at 4 degree, the next morning, add protein A-agarose IP reagent (20ul) from Santa Cruz (which should recognize IgG from the species the antibody is raised), incubate for another 4 hours at 4 degree.
- after the incubation, I centrifuged the mixture at 1000Xg for 5' at 4 degree, then washed with 1ml PBS buffer (cold) four times, each time, centrifuged as above. after last wash, added 25ul RIPA buffer and 2ul 20% SDS, boiled for 10'. then short spin and used ~13ul supernatant for WB, in which the primary antibody is the one from the other species.
With 20ug of the above lysate and the second antibody, I was able to see a lot of the protein of my interest by WB. The IP I described above used 5-times lysate as I used for direct WB. After IP, I loaded ~half of the supernatant. Theoretically I should see a fat band after WB of the IP samples. However, as other things in life that don't go theoretically, I saw a much fainter band. I also analyzed the supernatant after the first centrifugation before washing, there are still at least half that protein left behind.
I don't know what's wrong here. I can think of several possible reasons:
1) the overnight incubation. I taped the 1.5ml eppendorf tubes containing the lysate and antibody on a platform rotator. The volume of the whole mixture is no more than 100ul. Is it possible that the mixing was not complete? Should I switch to a vortexer with a tube holder, and vortex at a low setting?
2) The Santa Cruz instruction sheet for the IP reagent asks to incubate the antibody and lysate for 1 hour, and then add protein A-agarose IP reagent for overnight incubation. everything else is the same then. Does the difference between my procedure and Santa Cruz's account for the failure of my IP?
3) boiling step: I boiled for 10', following the protocol in our lab. But Santa Cruz's instruction sheet asks to boil for 2-3'. Does long-time boiling damage the protein of my interest?
I have been really frustrated by this issue recently. Any advice would be really appreciated! Thanks a lot!
please, to ensure getting any answer, summarize your question to the point