I have two identical proteins that were diluted from a stock but run on different days. There are slight differences in the spectra due to pipetting error and concentration differences because the absorbance spectra don't exactly line up perfectly. I calculated the ratio of the absorbances (higher conc/lower conc) and multiplied the CD spectra of the lower concentration species by this ratio. This definitely overlays the absorbance spectra but the problem is that even though the higher concentration species has higher absorbance values from 195-260nm, this doesn't necessarily translate for the CD spectra. There are places in the CD spectra that have a greater magnitude for the lower concentration species than the higher concentration species, which causes this absorbance ratio method to fail.
Does anyone have ideas on what the best way is to normalize two CD spectra of identical proteins that have slight concentration differences?
Thank you in advance.
The standard way to plot CD data is MRE ( mean residual elllipticity) as a function of wavelength. MRE accounts for concentration, thus normalizing it against concentration errors. All you need for MRE is the path length, concentration and number of amino acids in the protein. The equation do that is well known and I trust you can find it. :) Differences in concentration can result directly in amplitude of signals but should NOT change peak positions. If you see changes in peak positions, something went wrong like aggregation or precipitation.
Hi Rm, CD is at best a qualitative image of a proteins structural composition. It is not a quantitative measurement.
Hi Aditya Iyer ,
Thank you but I think you misunderstood the question. I converted to MRE and re-plotted the data and as I expected, the scale changes but the exact trend remains. The reason being is that you input the concentration. The proteins all were diluted to be at 2uM but because of pipetting error this is not the case and so you see slight differences in the spectra. Hope this makes sense.
Hi Rm, I love to be corrected when I am wrong.
That's what science is all about.
Post your spectra here if possible. Also double check if you are doing measurements in a Cl containing buffer. Cl ions absorb strongly in that range.
take a look on this paper, then download the tool provided by the author
Article 'CDtoolX, a Downloadable Software Package for Processing and...
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