What should be the maximum amount of DMSO used when taking CD spectra of protein to avoid the shooting up of the HT voltage. I used 0.1mm cuvette and 0.1%DMSO in my solutions and yet the HT was shooting up. I would prefer using 1%DMSO for my experiments otherwise. So any suggestions on this?
As you know the general norm is that a protein solution should contain the chemicals to maintain the protein stability/solubility and at the lowest concentrations possible. Any chemical product which absorbs in the region of interest should be avoided. In any case there are questions that can be resolved previously to discard a CD measure in the presence of DMSO.
The question is related to the region of interest of your CD measure. What is your region of interest? 160nm-250nm? 200-250nm? >250nm? etc… DMSO absorbs strongly in the UV. Thus, if you don’t need to measure at short wavelengths you will get a not bad CD spectrum in presence of DMSO. In any case you need to make proofs at different DMSO concentrations to see whether or not and in what degree your region of interest is affected by the presence of DMSO.
Also other things in your buffer can cause absorption in the UV, so it may nut just be DMSO. You can get the absorption of just a the buffer solution and adjust the concentrations to keep the HT at reasonable levels.
I have checked my buffer, it is a 10mM PBS which does not interfere with the spectrum. Currently I am using 0.1% DMSO. I cannot go lower than this as the solubility of polyphenols on which I am working on will pose another problem.As mentioned i have also reduced the cuvette pathlength to 0.1mm. As my protein lacks the aromatic amino acids , the CD signal given out is far less in 0.1mm cuvette which further demands a use f higher protein concentration for the signal which disturbs the balance in inter relating my data and other studies.
Because you have gone to nearly the lower practical limit of path length and co-solvent concentration with DMSO, I suggest you consider alternative solvents to solubilize the polyphenols, solvents that do not have such strong UV absorption. Acetonitrile, trimethylphosphate, and ethanol are possibilities.
Hi, hope it's still relevant. You should also change PBS to PB or MOPS. The Cl causes major absorbance disruptions in the ~190 nm region.
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