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Protein Purification: Column Chromatography - Ion exchange; Dialysis and Concentration

BCH5425 Molecular Biology and Biotechnology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber blaber@sb.fsu.edu Florida State University, adapted with permission.

Lecture 30


Column chromatography

After initial fractionation steps the typical procedure is to move to column chromatography.

  1. Ion exchange
  2. Hydrophobic
  3. Gel filtration
  4. Affinity

Ion exchange

Ion exchange resins contain charged groups.

Type of exchanger

Functional group

Common name

Weak cation exchanger

carboxymethyl

CM cellulose/sephadex

Strong cation exchanger

sulfopropyl

SP sephadex

Weak anion exchanger

diethylaminoethyl

DE cellulose/sephadex

Strong anion exchanger

quaternary amine

QAE sephadex

Generally speaking, ion exchange columns are short and fat in dimensions.

Elution of proteins from ion exchange resins

Note that after ion exchange chromatography the protein of interest will be in a buffer with a potentially high salt concentration. This must be taken into account before proceeding with the next step in the purification scheme

Note: often the buffer salt concentration is 0 M


Dialysis example

We have a 10ml protein sample from an ion exchange column elution pool which contains 1.0M NaCl. For our next step in the purification we can have no more than 1mM NaCl in the sample.

Therefore, the required buffer volume would be (total vol - sample vol) = 9.990 L (or ~ 10 L)


First dialysis versus 310 ml of buffer: sample NaCl conc will be (10*1.0)/(320) = 31 mM

Second dialysis versus 310 ml of buffer: sample NaCl conc will be (10*0.031)/(320) = 0.97 mM

Thus, instead of making 10 L of buffer, we could make only 620 ml and achieve the same results with two dialysis steps

A useful rule of thumb is that for most types of dialysis tubing the dialysis is 80% compete after four hours

Concentration

For both dialysis and concentration, it is essential that the membrane does not interact with the protein (i.e. has no affinity for, and will not bind, the protein)


1998 Dr. Michael Blaber


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