Why colorimetric assays for DNA and protein

[mansipan]

My concerns is, If DNA gives absorption at 260 nm and protein at 280 nm, then why colorimetric estimation is done for the nucleic acid and protein concentration determination, instead of just taking the OD at the respective lambda max and making the calculations

[CharonY]

Several reasons:

- in cell extracts there is a lot of other stuff that absorbs at UV wavelengths. If you need precise measurements that will mess things up, especially when concentrations are low

- the precise composition influences measurements. This is less of an issue for DNA, but for protein it is mostly aromatic amino acids that absorb at that wavelength. Changes in the ratio of aromatic to other amino acids are going to influence your measurements significantly

-dynamic range is often quite bad

 

In short, UV measurement just gives you a rough estimate. It works decent enough if you add separation of your compounds and with proper calibration (e.g. using HPLC-UV) but just using in a mixture e.g. using a photometer it will only work within a somewhat narrow range of concentrations and only with a well defined and purified sample. It will still be more imprecise, though.

[John Cuthber]

You can do colourimetric tests with relatively cheap equipment.

[mansipan]

Thanks for the reply.