Hi everyone,
I'm working with protein that is associated with short (few hundred bases) RNA fragments. In order to quantify the RNA per protein molecule, I took the absorbance of the sample at 260 nm and 280 nm. I have also isolated the total RNA and the total protein, and then quantified the RNA amounts and protein amounts. That gives me a slightly different ratio. (It is unlikely that there was loss of RNA during purification - I precipitated total RNA using guanidinium thiocyanate-phenol-chloroform extraction using TRIzol.)
Is it correct to rely on just A260/280 ratios for quantifying RNA/protein ratio; or is it preferable to use more direct methods like RNA isolation?
I agree with the comments from Abhijit, and would like to add some more information. When you measure the absorbance of your protein/RNA mixture solution at 260 and 280 nm, you evaluate just the average of all your proteins absorbance present in your solution. But if you need to quantitatively evaluate the relative abundance of your specific protein bound to your specific RNA(s) you need to know the extinction coefficient of your particular protein at 280 nm. You might want to use some RNAses to determine the amount of your protein at 280 nm, or you can use some recombinant (highly purified and RNAse free) proteolytic enzymes to determine the amount of your particular RNA(s) at 260 nm in your final protein/RNA solution.
If you are working with protein, then first try to treat your samples with RNAse to remove the RNA or else you can use Qubit based RNA or protein quantification kit which give you a accurate results.
I agree with the comments from Abhijit, and would like to add some more information. When you measure the absorbance of your protein/RNA mixture solution at 260 and 280 nm, you evaluate just the average of all your proteins absorbance present in your solution. But if you need to quantitatively evaluate the relative abundance of your specific protein bound to your specific RNA(s) you need to know the extinction coefficient of your particular protein at 280 nm. You might want to use some RNAses to determine the amount of your protein at 280 nm, or you can use some recombinant (highly purified and RNAse free) proteolytic enzymes to determine the amount of your particular RNA(s) at 260 nm in your final protein/RNA solution.
be careful : hydrolzing RNA or protein will solve your problem only if you get rid of the degradation products because these degradation products themselves absorb UV light in the same way as the undegraded product. In fact degradation of RNA will slightly increase the absorbtion.
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