Affinity Chromatography: A Two Step Method

BIOLOGY 371

INDEPENDENT RESEARCH

WEEK 3

Affinity Chromatography

A Two Step Procedure to Purify IDH

Preparation of a Crude Homogenate

[Perform all steps at 4° C]

1. Collect an appropriate amount of clean, fresh tissue - 200 to 400 gm should be adequate.
2. Homogenize this tissue in 2 - 3 volumes of cold homogenization buffer, using a [kitchen] blender.
3. Filter the homogenate through 2 layers of cheese cloth.
4. Put the filtrate into 50-mL plastic centrifuge tubes and store at -70° C.
5. When needed, thaw 2 - 6 tubes of homogenized tissue at room temperature, or overnight in the refrigerator.
6. Centrifuge the thawed material at 17,000 g for 15 minutes.
7. Determine the volume of the supernatant and save as the crude homogenate.
8. Save a small volume [0.5 mL] for enzyme assays and protein determinations.

Preparation of an Ammonium Sulfate Precipitate

1. Place the crude homogenate and a stir-bar in a beaker on a magnetic stirrer in the refrigerator.
2. Slowly add crystalline ammonium sulfate, while stirring, to make the solution 45% saturated [see Ammonium Sulfate Table].
3. Stir at least 15 minutes.
4. Centrifuge this material at 17,000 g for 15 minutes.
5. Discard the pellet [45% P].
6. Determine the volume of the supernatant and save as the 45% Supernatant.
7. Make the 45% supernatant 65% saturated with ammonium sulfate and stir for at least 15 minutes.
8. Centrifuge the 65% solution at 17,000 g for 15 minutes.
9. Resuspend the pellet [65% P] in a minimal volume of Affinity Chromatography Buffer [ACB].
10. Save a small volume [0.5 mL] for enzyme assays and protein determinations.

The resuspended pellet [65% P] still contains some ammonium sulfate, which will interfere with binding to affinity matrices. Thus, it will need to be "desalted". Desalting may be accomplished by dialysis or, more quickly, by gel filtration using Sephadex G-50 or G-75.

Matrex Red A Chromatography

1. Wash the Matrex Red A column with 50 mL of 8M urea.
2. Wash the column with 50 - 100 mL of ACB.
3. Pump the desalted fractions containing IDH activity over the column.
4. Wash the column with ACB until fractions are clear [or overnight, after setting pump speed at minimal setting].
5. Wash column with 150 mL gradient of 0 - 20 mM NADP and 0 - 24 mM isocitrate.
6. Assay for activity. Use 100 ul sample 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. Combine tubes with highest activity and concentrate using an ultrafiltration device.
8. 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.

Use gradient maker. Put 75 mL ACB on "outlet" side. Dissolve 33 mg of NADP⁺ and 420 mg of isocitrate in 75 mL of ACB and add to the "intake" side of the gradient maker. Attach the outlet tubing to the peristaltic pump and pump the gradient over the Matrex Red A column to elute IDH.

For large volumes, use the 50 mL unit and 25 - 30 pounds of pressure. The filter allows substances with molecular sizes smaller than 50K pass through. For smaller volumes use the 10-mL unit and no more than 25 pounds of pressure.

Table 1. Summary of purification of isocitrate dehydrogenase from Brassica hirta.

[Data of Toh Hean Ch'ng, Spring Semester, 1998.]

Material Volume [mL] Protein [mg/mL] Specific Activity* Fold Purification

Crude homogenate 249 5.52 0.1281 1

45-65% Pellet 11 9.57 1.302 10.16

Matrex Red, concentrated 1.35 0.256 69.48 542.4

µg of NADP+ reduced per min per mg of protein

From: Dye-Ligand Chromatography: Applications-Method-Theory of Matrex Gel Media. Amicon, Inc. 1993.

Figure 4. Purification of isocitrate dehydrogenase from Silene alba using Matrex Red chromatography.

Return to Schedule

Material	Volume [mL]	Protein [mg/mL]	Specific Activity*	Fold Purification
Crude homogenate	249	5.52	0.1281	1
45-65% Pellet	11	9.57	1.302	10.16
Matrex Red, concentrated	1.35	0.256	69.48	542.4