UV spectra of pure proteins A280 nm/A260 nm Experimental
UV spectra of pure proteins A280 nm/A260 nm Experimental
A relatively simple and effective measure of protein purity is obtained by determining the ultraviolet (UV) spectra of purified proteins. Even if sodium dodecyl sulfate (SDS) gel electrophoresis analysis shows that no heterogeneous proteins are present, the protein may contain mixed nucleic acids. A rough estimate of the amount of nucleic acid can be obtained from the absorption ratio of 280 nm to 260 nm. This experiment is from the guide to protein purification and characterization by Houzhu Zhu.
Operation method
UV spectra of pure proteins A280nm/A260nm experiments Move operating procedure For more product details, please visit Aladdin Scientific website.
1) Carefully measure the UV spectrum of the protein solution from 210nm to 340nm using a suitable buffer as a blank control.
2) If there is no absorption from 310mn to 340nm (protein does not absorb in this region), simply determine the absorption values at 280nm and 260nm, the A280nm/A260nm ratio should be 1.8~2.0.
3) If 310nm to 340nm can be seen obvious absorption, is due to light scattering (is due to protein large or aggregation), must be Leach and Sheraga (1960) method to correct the A280nm and A260nm value. This method plots the logarithm of the absorption value against the logarithm of the wavelength and extrapolates the absorption values from 310mn to 340rnn to determine the absorption values at 280nm and 260nm due to light scattering. The corrected absorption values at 280nm and 260nm can be used to determine the A280nm/A260nm ratio and to derive the protein concentration from E(1 mg/ml) (see P.168).