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· · · RNA Pictures 7 photos 7 comments |
· · · RNA Pictures 7 photos 7 comments | ||
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| Hi, My name is Jorge, I am trying to do some Coimmunoprecipitations of RNA with tagged-proteins. I am using the Ribonucleoside Vanadyl Complex, and in my hands it makes to precipitate RNA unespecifically. So, I have really nice CoIps, included the negative controls :-(, in which I obtained a High background due to this problem. Anybody has this problem? Anybody knows what is happening? or, what I can do? Thanks in advance, Jorge |
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| Thanks Koukou2 for help me, Yes I IP the proteins. I see them by Western blot and i do not detect background at all. The washes? I wash 5 times 5 minutes and 50 volumes each in a rotating wheel. My RNAs are from yeast and the cultives are chekced. I asses the RNA in Northern blot with specific probes. So I could say I have not contaminated RNA. I have tried several times with: Antibody plus gammabind sepharose Antibody-sepharose in both cases I have this unespecific precipitation. The buffer is prepared with the Ribonucleoside Vanadyl Comples at 52ÂșC to dissolve it completely and then filtered in 0.22microm. But during the precipitation, often I have some lumps that I think is what makes the trouble. I am sure that avoiding this lumps I will avoid the unespecific precipitation, but I do not how. And not I have not make RNA-EMSA, because I want to know wich rRNAs bind to a protein in different conditions. I wonder if anybody have use this RNAse inhibitor and could tell me how to use it, I would be very grateful. |