Affinity Chromatography: A three step method

BIOLOGY 371

INDEPENDENT RESEARCH

WEEK 4

Affinity Chromatography

Purification Of NADP-IDH By Affinity Chromatography

A Three Step procedure to Purify IDH to Apparent Homogeneity

After: Ni, W., E. F. Robertson and H. C. Reeves, 1987. Purification and
characterization of cytosolic NADP specific isocitrate dehydrogenase from
Pisum sativum . Plant Physiology 83: 785 - 788.

Prepare a 45 - 65% pellet as described in the previous section. De-salt this preparation.

DEAE Chromatography

All procedures are performed at 4° C and peristaltic pump speed set no higher than 2.
1. Wash the DEAE column with 50 mL of 8M urea.
2. Wash the column with 50 - 100 mL of ACB.
3. Pump the de-salted 45 - 65% pellet solution over the DEAE column.
4. Wash the column with ACB until fractions are clear.
5. Wash column with 150 mL gradient of 0.5 M KCl in ACB.
6. Assay 100 ul samples of every 3rd tube until activity is found.

Then assay every tube to define the peak. In peak tubes, 10 µl per assay may be appropriate.

7. Determine the optical density at 280 nm of the contents in an array of tubes to describe the protein "profile" of the material passed over the column.
8. Combine fractions with IDH activity. This material is ready for Matrex Red A chromatography [WEEK 3].

Figure 5. Elution of Brassica isocitrate dehydrogenase off DEAE column.

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