Hi Prakriti,

Firstly, the protein concentration of 10uM doesn't mean anything to me. In CD you are looking to the amide absorbance. Thus, without information on the sequence length of your protein, I can't know if 10uM is appropriate or not.

Then about HEPES buffer. If your question is : does HEPES buffer can distort the measured ellipticities ? I would say no. So far I know, HEPES is not chiral.

If your question is : does HEPES buffer can decrease the signal/noise ratio ? Then the answer is yes. Higher the salt & buffer concentration, lower the signal/noise ratio. That's the reason why you probably already heard that in CD we have to decrease salt as much as possible and use a phosphate buffer. The latter doesn't absorb in the wavelength range we use for protein secondary structure characterization, it has no impact on signal/noise ratio. TRIS is not really good as it also absorb.

But ! the decrease of signal/noise is due to absorbance and thanks to Beer-Lambert we know that it is proportional to the particles concentration and the light path. Thus, if you really don't want to change your buffer composition, you can still use an other cuvette with a lower distance. However, you will need to increase your protein concentration by the same factor.

Good luck

Antoine.

Circular dichroism is usually measured in the far UV, down to 180 nm with conventional and to 140 nm with special equipment. Your buffer should absorb as little as possible at those wavelengths, and also show no photolysis. Therefore, 10 mM phosphate is commonly used. Perchlorate, sulphate or fluoride is used to adjust ionic strength.

Hi!

Thanks for your suggestions. I was able to get good CD data by using 5 mM phosphate along with 10 mM NaCl buffer. The dynode voltage was a maximum of 530 V.