i was trying to take far UV spectra of a protein sample (buffer: 10 mM NaPi, 100mM NaCl, 2mM DTT). Till 210 nm there is no noise in the spectra but in the range 190 to 210nm there is lots of noice and even the voltage is more than 1000 V. I have to use DTT in the sample, since the protein is dimeric in nature.
As Michael Schmitt said, the sample is not sufficiently transparent in the 190-210 nm range, as evidenced by the high voltage. The solution, if you can't change the buffer composition, is to use a higher protein concentration and a shorter pathlength cuvette.
If DTT is causing the high absorbance, maybe you can replace it with a different reducing agent that causes less absorbance (TCEP, 2-mercaptoethanol?), or just leave it out for the CD measurement. Formation of a disulfide bond may not affect the secondary structure.
The other method to prevent disulfide formation is to block the sulfhydryls with N-ethylmaleimide or iodoacetamide.
For me this looks like sufficient light intensity is not measured = sample is not sufficiently transparent (small denominator can lead to large amplitudes). You might check this by measuring a UV-(vis) of your sample (and a working one). The equipment is able to result in valid data in this range?
As Michael Schmitt said, sample turbidity can be an issue. Do you have the same issue with your blank (just buffer)?
What size cuvette are you using? If you are able to use a thinner cuvette (0.5 -1 mm) you might be able to work around any turbidity. Or you can try dilluting with your buffer as long as the blank is ok.
Mat
As Michael Schmitt said, the sample is not sufficiently transparent in the 190-210 nm range, as evidenced by the high voltage. The solution, if you can't change the buffer composition, is to use a higher protein concentration and a shorter pathlength cuvette.
If DTT is causing the high absorbance, maybe you can replace it with a different reducing agent that causes less absorbance (TCEP, 2-mercaptoethanol?), or just leave it out for the CD measurement. Formation of a disulfide bond may not affect the secondary structure.
The other method to prevent disulfide formation is to block the sulfhydryls with N-ethylmaleimide or iodoacetamide.
thank you all for help,
Mathew Sebastiao I have used 2mm cuvette, and even the buffer is giving the noise.
Adam B Shapiro
I am using DTT because, after purification, during dialysis step protein gets oxidised and it starts to precipitate or aggregate. I can clearly see visible small white particles in the sample after dialysis, if i have not added DTT.
There are much thinner CD cuvettes than 2 mm. They consist of two separate pieces with ground glass edges. These make it possible to collect data to lower wavelengths by reducing the buffer absorbance. The protein concentration should be increased, of course, but a smaller volume of the protein is needed. You can concentrate the protein quickly using a centrifugal ultrafiltration device, if necessary.
The buffer you are using will absorb massively between 190 and 210 nm. Do you really need that much NaCl? I would use ten times less or none at all. We also use 0.5 mm cuvettes for far-UV CD and avoid phosphate buffer.
Buffer alone has not shown drastic noise its very little in the range 200-260.
if the buffer alone do not show noise, just try to reduce your sample concentration.
i have used 20 micro molar protein solution (10mM phosphate buffer with pH 6.6, 100 mM NaCl, 2mM DTT) (pI of protein is 8.93, and the molecular weight is 30.9 kDa)
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