I have GST tagged purified recombinant proteins and would like to go for CD analysis. Does someone have the sample preparation protocol for running CD analysis?
Also, can I use GST tagged protein for CD?
An important consideration is the composition of the buffer. Many common buffer components absorb very strongly in the far-UV and thereby interfere with CD. Here is a web page that will help you to select a suitable buffer:
http://structbio.vanderbilt.edu/wetlab/cd.sample.prep.php
You will also have to optimize the protein concentration to get a strong signal but not cause too much absorbance.
Very short pathlength cuvettes (1 mm or less) are preferred for CD to minimize the absorbance by the buffer. Make sure the cuvettes are perfectly clean.
Dear Adam,
Thank you very much for your information. Can I use GST tagged protein for CD? mean to say with out cleaving the GST tag form protein.
Thanks,
Swapnil
Unfortunately you can't use GST tagged protein cause your final spectra will be the sum of your protein plus GST.
With CD you can use as little as 10ug/ml of protein but it is sensitive to salt, you should work with max 50mM NaCl with a 1mm cuvette, alternatively you can use 0.1mm one and the use up to 150mM.
Anyway all details shall be revealed in the attached protocol:
I can highly recommend this paper for CD spectroscopy
Article How to study proteins by circular dichroism
Many GST tags are cleavable. What about yours? Can you cleave off the GST tag and use the pure protein for CD spectroscopy?
What is the reason why you would prefer doing CD spectroscopy with the GST tagged protein?
Hello Heide,
Thanks.
No I don't have ant preference for GST tagged protein. I just wanted to gain the information. Yes, now I have got the idea about cleavable GST.
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