Doubt regarding protein dialysis and r-protein purification????

sudheer sangeetham via methods%40net.bio.net (by sudheer.pbm07 from gmail.com)
Sat Jun 23 11:22:24 EST 2012


Hello Everybody
Hope every research is going well. I would like to ask you 2 doubts

1. Regarding Protein dialyis: I want my purified protein in 1xPBS. So after
purify the protein,15ml I dialysed the protein against MQ water,5000ml for
1 hour at room temperature then I dialysed the protein in 1x PBS,5000ml at
kept at 4 degree for overnight. The next day morning I collected the
samples. I changed the dialysed buffer only once, If I do this way the
purified protein is free of imidazole and the protein properly soluble in
1xPBS??? I did this way because I dont want use much PBS ?


2. The recombinant protein purification:  The protein , which I am
purifying most of the protein bind to Ni-NTA though the purification
process was finished. I could say 70% protein I am getting in elution
fractions remaining protein still stick to the beads. I wanted to reuse the
columns for many times. So Could anyone please suggest any buffer, which
can wash and elute the protein from the bead???? So I can reuse the column.

If anyone answer me for my questions that would great help to me from them.

Thank you very much in advance

Cheers

Sudheer


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