Dear Shubhmita,

Tris is not a good buffer for CD especially at these low wavelengths. Use 25 mM NaPhosphate instead. The other thing you can do is to increase your protein concentration (~10x) and organize a 0.1 mm quartz cell. It also helps to accumulate more scans for averaging.

For more information:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728378/

I hope this helps.

Best regards, Alex

Thanks Alexander. 

I will try with sodium phosphate and 0.1 mm quartz cell. Can you tell me how increasing the protein concentration will help? I cannot increase the concentration for few samples, the concentrations however are above 0.2 mg/ml. 

Will let you know how things go.

Best,

Shubhmita

The Vicente's suggestion is a very good one, but you must change completely the buffer (for. ex. by dialysis), by using a higher protein concentration and, at same time,  reducing as much as possible the optical path. Remember also that you are operating without buffering the solution, thus you must perform the measurements very rapidly using as solvent a salt solution (Na or K fluoride) previously purged with N2 (spectroscopic grade) in order to dampen pH change due to CO2, because the solution is now very sensitive to pH changes induced by environmental CO2. Look the sequence of your protein, compute its net charge from aa, if the value is between -1 and +1 at neutral pH, then the probability that a pH change may affect the protein conformation is low.

I hope this is useful

replace Tris with phosphate buffer and NaCl with sodium sulfate. u will get proper data

dialyse properly before experiment with this buffer

Thanks Vincente, Giovanni and Timir. I have started working with Potassium phosphate and sodium sulphate buffer. Working really well. Thank you!