Low protein yield with immunoprecipitation - (Nov/08/2006 )
Hey,
I have been struggling with my pulse- chase experiment for quite a long while. In short, I expose my cells (CHO) with tritium for one hour followed by lysing the cell in RIPA buffer. Immunoprecipitation (incubation is being done overnight) is done with Anti-xPress antibody (mouse IgG1) from invitrogen (protein of interest, ACAT2 is tagged with Anti-xPress). Upon immunoprecitation, I elute the protein from the beads (Protein G-Agarose beads from Calbiochem) either by heating at 95 degrees or leaving it at 37 degrees for 30 minutes. SDS-PAGE follows by fluorography is performed using the eluted protein.
Recently, I did a bradford to quantify the amount of immunoprecipitated protein and realised that the concentration is 1/62 of the whole cell lysate (ACAT2 is expressed in very amount in the cell, does that explains this?). I supposed this explain why I did not get any results after exposing my film for a month. Is there anyone who can suggest any alternations I can make to my protocol? Are there any problems with the elution conditions? Preferably, I prefer not to heat the bead as ACAT2 is prone to aggregation upon heating, is there another way around this problem. Thanks!!
I have been struggling with my pulse- chase experiment for quite a long while. In short, I expose my cells (CHO) with tritium for one hour followed by lysing the cell in RIPA buffer. Immunoprecipitation (incubation is being done overnight) is done with Anti-xPress antibody (mouse IgG1) from invitrogen (protein of interest, ACAT2 is tagged with Anti-xPress). Upon immunoprecitation, I elute the protein from the beads (Protein G-Agarose beads from Calbiochem) either by heating at 95 degrees or leaving it at 37 degrees for 30 minutes. SDS-PAGE follows by fluorography is performed using the eluted protein.
Recently, I did a bradford to quantify the amount of immunoprecipitated protein and realised that the concentration is 1/62 of the whole cell lysate (ACAT2 is expressed in very amount in the cell, does that explains this?). I supposed this explain why I did not get any results after exposing my film for a month. Is there anyone who can suggest any alternations I can make to my protocol? Are there any problems with the elution conditions? Preferably, I prefer not to heat the bead as ACAT2 is prone to aggregation upon heating, is there another way around this problem. Thanks!!
amount of precipitated protein depends on the cellular abundancy of your protein of interest, and the composition of the complexes which are co-precipitated as well as on specificity of the primary antibody and its amount which was used; some protocols are too optimistic and recommend only some µl of Ab; increase the amount of Ab for IP, and try to diminish the washing of the imminoprecipitate
immunoprecipitation problem, or elution problem.
normally after boiling 5 min at 95 it should be eluted, but if it forms aggregate, it will not be OK.
I don't know if 30 min at 37°C is efficient.
there is also the acidic elution. have a look on the forum, there are several topics about it.
(sorry, I don't have many time right now)
I'm not very used with the tritium.
Have you ever tried to read in a scintillator counter the amount of radiactivity in your beads, before and after elution, in the supernatant and in the beads? (I don't know if you have enough radioactiity to count it)
Thanks, I didn't thought about doing scintillation counting with the bead, I will try it next week. I will keep you guys posted.