My protein was present in 50 mM Sodium Phosphate, 500 mM NaCl and 10 mM Imidazole. It was loaded onto Ni-NTA and washed with same buffer but 20 mM imidazole. Then the protein was eluted using 50 mM Sodium Phosphate, 500 mM NaCl and eluted from a gradient of 0-500 mM Imidazole (eluted at ~250 mM imidazole conc.). So now my question is do I need to remove both imidazole and NaCl from it.
Hi Mangal Singh
It depends on the purity of your elution and the application of the product. If you want to purify it by some ion exchanger that case you need to remove the salts to bind it, if you choose HIC then you can directly bind it or you can add some more salt to increase binding.
If your elution is pure and IMAC is the final step in purification then you need to remove the imidazole, but NaCl is acceptable upto 150mM for formulations.
Good luck.
Hi there,
It is always better to have the protein sample composition in hands. Furthermore I guess the imidazole present of the elution fraction is of no use for downstream applications. So what I would do to get rid of it is to run SEC in the buffer the protein likes the most. The other good thing about SEC is that it will give you information about oligomerization state of your protein
On the contrary if your protein is happy with the 0.5M NaCl, there is no need to eliminate it unless as mentioned above it would interfere with downstream experiments...
Hi Mangal,
It all depends on what you are going to be doing with the protein, post purification; if it is proteomics, the easiest way to "purify" your protein, free of additives, is to carry out SDS-PAGE. Not only does this step desalt the protein, it also denatures and readies the protein for in-gel digestion.
Hediye
I have to use the protein further for enzyme assays. I have desalted the protein in 50 mM Sodium Phosphate buffer pH7, containing 50 mM NaCl using PD-10 Desalting columns. I hope the protein will work. Thanks all for your valuable suggestions.
Hi,it all depends on your downstream purpose,
It all depends on your downstream purpose.
But, if you have large amount of proteins and your usage is less for your analysis, the rest quantity of proteins can be monitored with different enrichment methods(desalting or lyophilization and stored the proteins in suitable for long storage native or denature)
goodluck!!
Nickel will also leach from the column and might affect the activity of your protein or your downstream use.
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