For a pure protein, the A260/A280 ratio should be 0.5-0.55; higher values suggest nucleic acid contamination. Nucleic acids will also lead to an overestimation of the protein concentration. If you use metal affinity chromatography purification, you may try to get rid of them by performing a high salt wash, e.g. 4.5 M NaCl, if your protein can tolerate it. Then, return to a lower salt wash buffer before proceeding to elution with imidazole.

Good luck,

Pierre

Dear Kokouvi, I second Pierre.

Additionally, presence of aromatic amino acids sometimes results in higher A260/280 ratio. Thus, the ratio higher than 0.7 indicates nucleic acid contamination.

This ratio depends strongly on the nature of the protein (how many aromatic aminoacids, the secondary/higher order structures. etc), it will vary between the proteins. The best would be to produce a pure, structured protein and make a standard curve, if this is possible! Good luck!

Dear Pierre, Our targets are recombinant proteins carrying a His tag, so we use metal affinity chromatography matrix (Ni-NTA Agarose) for purification in our lab. As suggested, we will try to get rid of contamination at earlier/middle stages of the purification before final elutions with imidazole.

Dear Amit, We have obtained a range of different ratios from different purified antigen targets, of which few are above 0.7. So, these suggested that there may have both aromatic amino acid and nucleic acid contamination in our samples.

Dear Mariana, The proteins were shown to be stable. We also consider quantifying the antigen targets using BCA protein assay.

Thank you all for your expert clarifications and suggestions.