I purified two ~ 55KDa proteins having C-terminal His-Tag. The concentration of both the proteins are ~ 40 uM. One is the wild type and the another is the mutant protein. The proteins always elute with some non-secific bands which are very faint as visible on SDS PAGE (image is attached). I am looking forward to perform Circular Dichroism of the proteins.
Can I perform CD with these proteins containing faint non-specific bands?
Hi
I absolutely agree, your protein sample is suitable for the CD experiments. This lets say 10% impurities, are smaller fragments as I can see (from the gel) and for sure will contribute much less to the Far-UV CD signal (as in the CD signal what counts is the quantity of the peptide-bonds).
However for more precise measurements, especially if you do thermal/chemical unfolding monitored by CD then is better to have as purest as possible protein. If you are characterizing this protein for the first time and at the same time you have opportunity to do LC-MassSpec analysis on your samples I would strongly recommend it. Then you will have more accurate data of the sample purity and most probably you will know if the small bands are coming form the degradation of your protein
good luck
It seems that your purified protein is 90% pure. I guess you would have very little influence of those contaminant proteins since your desired protein occupies approximately 90% of the molecules.
You may have to check whether these bands are contaminants or simply degradation of the same protein product.
Hi there,
You proteins are pure enough for CD. Just run the experiment and you will see! The critical point is the folding of the proteins of interest. Are they biologically active and/or do they run according to their expected size in SEC?
Hi,
Your protein seems ~90% pure. You can definitely carry out your CD experiments. It seems highly unlikely that they will interfere with your CD signals.
All the best
Hi
I absolutely agree, your protein sample is suitable for the CD experiments. This lets say 10% impurities, are smaller fragments as I can see (from the gel) and for sure will contribute much less to the Far-UV CD signal (as in the CD signal what counts is the quantity of the peptide-bonds).
However for more precise measurements, especially if you do thermal/chemical unfolding monitored by CD then is better to have as purest as possible protein. If you are characterizing this protein for the first time and at the same time you have opportunity to do LC-MassSpec analysis on your samples I would strongly recommend it. Then you will have more accurate data of the sample purity and most probably you will know if the small bands are coming form the degradation of your protein
good luck
Dear Sagarika,
yes, also from my experience you certainly can perform CD stectra analysis with these proteins. They are very pure, so do not worry.
Be sure spin your proteins at 4-6 degrees for cca 10-15 min. at 13 000-15000 rpm. just before the measurement to remove any potential aggregation.
Good luck!
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