Dear all,
I'm working with protein purification, the protein has a theoretical pI of 5.95 along with TRX tag. can any one suggest me which buffer has to use for maximum protein elution with Ni-NTA coulmn.? If possible send the reference article links.
Thank you
Why do you want to use Ni-NTA if you have thioredoxin tag?
Here: http://oxfordgenetics.com/dna-component-information/peptide-tag-options/thioredoxin-tag-plasmid-vector-information
they write that thioredoxin-tagged proteins are often stable up to 80°C. That would be nice pre-purification step. Also affinity chromatographies should be available for TRX-tags.
Concerning your pI, you should be at least one unit away from pI, thus the pH of your buffers should be more than 7 (or less than 5). But you really have to find, what works best for you.
Tris Hcl 20-50 mM. it has a pka of 8.1 so you can obtain a buffer at pH 7.3 that is perfect if you are purifying a human protein.
Well Tomas Hluska.....its very useful information for people searching for protein purification with TRX tag which itself is stable up to 80°C.
Hi Boya,
I am assuming you use TRX tag in the aim to facilitate protein crystallization, and in the tag there is a His tag sequence attached to the N-terminus of thioredoxin.
I think the principle of choosing buffer is to keep away from the pI of your protein and maintain a reductive environment for thioredoxin, thus I'll recommend 20-50 mM Tris-HCl pH 7.0-7.5, 100-150 mM NaCl, 5 mM β-mercaptoethanol. You can work out optimal pH and salt concentration in your own experiment.
Here I also attached a reference article:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2590909/
Good luck!
thank you Wenjiang Ma for your suggestion. I will definitelu work on this. thank you once again.
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