Is SDS going to interfere with the CD or fluorescence signal?
Any suggestions/experiences?
Regards.
Hi Mohammad, are you looking at Trp fluorescence? There are reports that SDS causes a blue shift in Trp fluorescence:
http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=5&cad=rja&uact=8&ved=0CEEQFjAE&url=http%3A%2F%2Fwww.mdpi.com%2F1422-0067%2F13%2F3%2F3229%2Fpdf&ei=ZHxrVP3CD421sQTvVg&usg=AFQjCNG9UIVtH_qukgo8q5e_PZXQ_6reHg
But then again, most denaturing reagents cause a Trp blue shift.
Hi Mohammad! In my experience SDS does not interfere with the CD measurements.We did check the denaturation behaviour of Beta Lactoglobulin at 4% SDS (typically in the SDS-PAGE buffers) and did not have any problems. However just to put a note here that if you are also looking to test the spectra in the presence of a reducing agent such as DTT, the case may be different. DTT has strong absorbance at 280 nm when analyzed by spectrophotometry. I would suggest you to take scans of your components individualy to see if theyw ould interfere with the method. Do share your findings if possible! Good luck.
Are you trying to do a protein stability study by titration of SDS or confirm an unfolded state in SDS? The problem with using SDS in a stability study is, unlike GuHCl and urea, the denaturing power of SDS is nonlinear. Even above the CMC, SDS micelles can vary in size and shape with SDS concentration and other factors like ionic strength. The results may become difficult to interpret.
If you are just confirming an unfolded state in SDS, the results will probably be qualitative rather than quantitative since I don't think you can assume the protein is 100% random coil even in high SDS concentrations.
I agree with Jeffrey, SDS can be used as protein denaturing agent but the analysis of the results is not simple. In addition with the concentration dependent changes in SDS structure, hydrophobic clusters of SDS molecules are also able to induce helical structures in amphipathic non-structured chains. Thus, the final denatured state has high content of secondary structure.
The global shape of protein-SDS complexes was first described by Reynolds and Tanford as rod-like prolate elipsoids. More recently, these complexes were modeled using data from NMR, light scattering, fluorescence spectroscopy and small angle X-ray scattering showing structures denoted as decorated micelles (with the unfolded protein wrapped around detergent micelles) and pearl necklace or necklace-and-beads structures (micelles on several parts of the unfolded protein chain, randomly decorating the polypeptide backbone).
In my experience, total fluorescence intensity is the best signal for monitoring SDS induced protein unfolding.
SDS is not that good denaturant like GuHCl or Urea. Although SDS has no known effect on CD/ fluorescence studies, but better to use controls. you should avoid studies higher than critical miceller concentration (CMC) or you will end up with ambiguous results. You may try a detergent whose CMC value is quite high. chemicals like DMSO, Imidizole etc interferes in CD studies.
There are published papers where SDS has been used to study denaturation and amyloid formation using CD and fluorescence. For example: Khan et al (2012) PLoS ONE Vol. 7 No.1
But as others have suggested that use urea/ Gdn HCl to study protein denaturation.
Urea or guanidine hydrochloride are the first choice for protein stability studies.
However, some proteins (e.g. helical membrane proteins) are resistant to chemical denaturation by these chaotropes or, in some instances, they lead to irreversible unfolding.
Ionic detergents such as SDS have been efficiently used to unfold some of these proteins under equilibrium conditions.But as was mentioned, experimental design and data analysis have to be performed with caution.
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