hi everyone,I am trying to purify a His Tag protein by passing it over the nickel column but too much of the protein actually flows thru and does not bind. there is also non specific binding as i see too many bands in my washings. i am using 10mM imidazole, 8M urea nad 20mM Tris as washing buffer and 500 mM imidazole, 8M urea and 20 mM Tris in my elution buffer. Can anyone plaease tell me about the volume of Ni-NTA to be used for binding a specific volume of proteins in a solution. I would appreciate any ideas and any possible soliutions thanks and regardswaji
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