ChIP assay loading control - (Mar/09/2005 )
I am planning to do Chromatin immunoprecipitation, but i am confused, i do not know which one is better for the loading control? start the same number of cells per condition and use 1/10 amount to be the loading control OR count the protein concentration and use same amount of protein ,then extract the DNA to do the PCR .
I think the first way is just the start matteral is same , but can not tell the process. The second way is start the same protein for IP , I do not know, somebody help me?
Yes, first use equal number of cells. You can do this by using duplicate treatment dishes, one dish for ChIP and the other solely for cell counting.
The second control is to set aside input DNA (1/100) after adding the ChIp dilution buffer but before adding ChIP antibody. When doing ChIP PCR, amplify the input DNA as well as ChIPed DNA. the intensity of the input DNA amplification will tell you the amount of starting DNA.
Another control for subsequent steps such as those multiple washings is to amplify some control gene. For example, if you are studying gene activation by histone acetylatio, you can amplify the G3PDH gene because its promoter is supposed to be acetylated. But if you are studying some repressive histone modifications, it is hard to control because the result is absense of amplicon.
Thanks , pcrman. really helpful. Just one thing , i am not sure: even I start the same number of cells , the input DNA still can not be the same and the amount of protein to do IP is also not same , so what can I do to start the chip assay , the same amound of cell number or the same amound of protein ?
Thanks a lot! wll
Thanks a lot ! but one thing i am not quite understood is even I start with the same amount of cells , the strat protein amount for IP maybe not same , so I will start the same amount of cells to continue teh rest of steps or start the cross linking and start the same amound of protein ,then continue the rest steps?
Thousands of thanks!
As far as I know, you don't need to quantitate the starting amount of protein. As I said, you just need to start with the same amount of cells and use control gene. That should be enough.
I see, thousands of thanks! I really appreciate it!
Another alternative I haven't seen anyone mention here is to quantitate the amount of diluted input DNA per sample (the same stuff you'll use for input sample PCR). I routinely use Hoechst 33258 to quantify such small amounts of DNA. This is essentially very similar to determining protein concentration - perhaps a bit shorter. I would caution against only using the same # of cells and believing that the PCR results are quantitative. Small deviations here will be amplified by the PCR portion of the experiment. A (+) control gene is good too, but there's no way to go back and fix the actual samples if the (+) control bands aren't exactly the same. Perhaps both methods would be best.
I believe DNA quantitation is the most accurate way to normalize samples for PCR. Add the appropriate amount of enriched DNA for PCR and your results can be compared on a gel.