Hey everyone,
I have purified GST-tagged protein in elution buffer (200 mM Tris-HCl pH 8, 20 mM NaCl,
40 mM reduced glutathione, 10 % Glycerol)
I read that the glycerol can be problematic for BCA assay, so is there a better method for measuring the concentration of my recombinant protein?
Thanks in advance!
Ensure that your protein is sufficiently pure. Use A280 is the best method but also;
1. Ensure that your protein has no contaminants (DNA, RNA or other proteins) that can skew the absorbance readings. If you can be assured that your protein is 99% pure then that's great.
2. Make sure that your zero controls for buffer components exactly as they would be in your protein (e.g. if you have imidazole or reducing agents.
3. If your spec can handle it take a scan from 180-300 nm. You should get a peak around 280 with buffer 'flare' at the far UV. If you have a very wide peak spanning the 260-250 region then you have DNA/RNA contamination.
4. Take readings for different dilutions of your protein (absorbance is accurate within a narrow range so you'll get inaccurate results at too low and too high protein concentration).
This was the best answer provided by a similar question in 2013. The thread is given for detail: https://www.researchgate.net/post/Quantification-of-recombinant-protein
Christina Czada Will you measure sample concentration without dilution? I wonder it will be out of appropriate measuring range when your sample has high concentration. How about dilution by appropriate buffer without glycerol in advance?
Why you are adding glycerol to elution buffer? Is it possible to exclude or reduce the concentration of it?
By the way, as Michitoshi Watanabe indicated if your sample conc. is allowing you to dilute your sample you may choose either BCA or Bradford assay.
Additionally, 1–10 mM DTT can be included in the binding and elution buffer.
There is no "best" method, Christina. In order to obtain comparable results, one should use the same method for all steps of the experiment.
Different proteins will give different results with different methods, as all methods are utilizing different properties of proteins.
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