Hello. I am a graduate student making recombinant proteins at university.
Recently, I have succeeded in the expression of recombinant proteins and purification and refolding using Ni-NTA, and eluted by elute buffer containing 500mM imidazole . How can i quantify proteins eluted by 500mM imidazole?
There is a less accurate way using a spectrophotometer such as a NanoDrop, where you use your elution buffer to blank it, and using the extinction coefficient and molecular weight of your protein, you can get the approximate concentration of your protein.
The other option (which is more accurate) is a method using BCA assay, there are protein quantification kits for this such as:
https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf
However, you might have to dilute your protein in Imidazole-free buffer because BCA agent tends to be sensitive to imidazole concentrations this high.
You can try to dialyse the imidazole out before performing the BCA assay, which will probably give you better results.
The Bradford assay is compatible with 0.2 M imidazole, so a bit of dilution of your purified protein will get you into range. Be sure to include the same amount of imidazole in the standards. Or dialyze out the imidazole (but people have mentioned that their protein precipitates when they do that, so be careful.)
Most proteins elute well from Ni-NTA with 0.25 M imidazole, so you may not need to use 0.5 M, and diluting from 0.25 M could also bring it well below the 0.2 M range mentioned by Adam. Aside from dialyzing, you can pass the protein through a buffer exchange column like PD-10 to remove the imidazole (if it's not amenable to a long stint of dialysis).
Another way to reduce the imidazole concentration is to concentrate the protein using a centrifugal ultrafiltration device, then redilute it with imidazole-free buffer.
A good way would be to concentrate the protein using a centrifugal concentrator to 1ml and then dilute back to 10ml in buffer devoid of imidazole. Reconcentrate to 1ml and again dilute back to 10ml of the previous imidazole-free buffer. This would ensure a 100-fold buffer exchange [in principle your protein should now have ~5mM imidazole] and now using a spectrophotometer (like nanodrop where you need minute samples) you can measure the absorbance at 280nm and from the extinction coefficient and molecular weight of your protein, estimate the protein concentration quite accurately.
Dear Min Cheol Kim
If your eluted protein has a good concentration you can use colorimetric methods as Bradford or BCA since you can dilute the sample and therefore reduce the imidazole concetration to perfrom it.
If you are using high purity imidazole (which show low absorbance at 280nm) you can also use the UV(280nm) determination.
https://proteocool.blogspot.com/2021/07/tips-of-week9-use-of-low-purity.html
Otherwise if you protein is diluted you can perform a rapid buffer exchange in a imidazole free buffer using desalting (eg PD-10 gravity flow colomn) and obtain a more precise quantification in a imidazole free buffer.
https://www.blogger.com/video.g?token=AD6v5dwNlVHz3BclDT9RLY-6KbEEqkS3RnRdHn4BqTsZW4j_prf3KZwHDMTzRYT-B9Yifvmde7RdExL5AArLFtg1Q13fu4-83o3M8bCka1jJ5PfWFK8B53BOpx6YrzvLr2qDiPgT15c
good luck
Manuele
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