protein purification column

Shaun D. Black SHAUN%JASON.DECNET at relay.the.net
Fri Mar 4 22:41:34 EST 1994


If your protein has a pI of 4.5, then it will have a preponderance
of acidic amino acids, relative to basic residues.  That is, Asp
and Glu will be present in higher percentage than His, Lys, and Arg.
At neutral pH, such a protein will have a significant negative
charge, and would wash right through a cation exchange column.  If
that is what your aim is, proceed ahead.  But, a wiser strategy
would be to take advantage of the strongly acidic character of your
protein and attempt separation on an anion exchange resin.  Numerous
possibilities exist: Traditional DEAE-cellulose phases (e.g. from
Whatman), Fractogel-DEAE (EM), Mono-Q (Pharmacia), MemSep (Amicon),
DEAE-Sepharose-Cl (Sigma), etc.

A general procedure to use for your protein on an anion exchanger
might be:
    -Read the mEq/ml rating on the stationary phase bottle. Use this
     to decided how much to use when pouring your column. Typically, 
     pour enough stationary phase that would bind about 2x your
     desired protein loading.  Going higher than this will risk low
     yeilds, and going lower than this risks poor separations or
     wash-through. 

    -Pour your column and equilibrate it with a minimum of 5 column
     volumes of 10-20 mM buffer, pH 6-8 would be OK.  I would
     choose Tris or Phosphate, probably.  Include protease in-
     hibitors and/or preservatives if you see fit.  Some proteins
     also require stabilizers such as substrates, ligands, glycerol,
     sucrose, etc.

    -Apply your sample at an ionic strength similar to what you
     used to equilibrate the column.

    -Elute with a gradient.  I've found that most proteins elute
     best with a salt gradient.  NaCl, KCl, KPO4 all work well.
     The gradient would need to go to 1-2M NaCl or KCl, but 0.5-1
     M would probably work for KPO4.  Plan for the gradient to be
     about 2-5 column volumes, depending on the 'steepness' that
     you desire.

    -Collect fractions equivalent to about 1/20th of the column
     volume.  Adjust size if your peaks are sharper or broader.

    -Assay fractions for activity or run SDS-PAGE to see where it
     has gone.

Hope this is helpful to you.  Cheers,  Shaun
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= Shaun D. Black, PhD  | Internet:shaun%jason.decnet at relay.the.net= 
= Dept. of Biochemistry| University of TX Health Center, at Tyler = 
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