Process Separations

* Refer to Bulletin 3193 for purification conditions.

Affi-Gel Blue Affinity Chromatography Gel for Enzyme and Blood Protein Purifications

Structure of Affi-Gel blue gel

Affi-Gel blue gel is supplied ready to use as an aqueous slurry of fully hydrated gel. It is available in two convenient particle sizes: a faster flowing 50–100 mesh (150–300 µm) and a slower flowing 100–200 mesh (75–150 µm). The gel is also available in convenient Econo-Pac cartridges, which can be used with the Econo system, FPLC, and HLPC systems.

Comparison of Cibacron Blue Affinity-Based Formats for Depletion of Albumin From Samples Prior to Two-Dimensional Gel Electrophoresis

Affi-Gel heparin gel is a ready-to-use affinity support for the purification of a wide range of proteins. It consists of heparin coupled via amide bonds to crosslinked agarose beads. As a result of this coupling, the gel is stable in all commonly used buffers and denaturants. No loss of activity is apparent after multiple cycles of use and regeneration. Affi-Gel heparin gel contains 1.0 (±0.2) mg heparin/ml gel, and will bind at least 1.2 mg human antithrombin III/ml gel. It is supplied hydrated in PBS, with 0.02% sodium azide as a preservative.

Heparin is a linear glycosaminoglycan, composed of mostly sulfated 1–4 linked glucosamine and glucuronic acid residues. It binds a variety of enzymes and other proteins, either ionically or by a more specific enzyme-inhibitor (or enzyme-activator) interaction. Heparin binding has been demonstrated in such protein classes as coagulation factors and other plasma proteins, polynucleotide polymerase, nuclease, endonuclease, lipase, lipoproteins, and proteases. These, and other similar proteins, may lend themselves to affinity purification on Affi-Gel heparin gel.

For an extensive review of heparin-agarose affinity chromatography, see Reference 1. Reference 2 reviews heparin-protein interactions.

Instructions for Use

1. Pour column of Affi-Gel heparin gel. (The capacity of the gel for various proteins is typically 0.2–2.0 mg/ml gel.)
2. Equilibrate the column with 3–5 bed volumes of the application buffer (a common buffer is PBS, 0.01 M KH₂PO₄, 0.15 M NaCl, pH 7.4).
3. Apply the sample in application buffer at a linear flow rate of 10–20 cm/hr.
4. Wash the column with application buffer until the O.D.₂₈₀ nm of the eluant is the same as the application buffer.
5. Elute the bound protein with application buffer plus 1.5 M NaCl.
   When more than one protein binds to the column, resolution may be achieved by a linear gradient of 0.15–2.0 M NaCl in the application buffer, or by stepwise elution with increasing concentrations of salt in the application buffer. Elution with heparin (at a concentration of 1–10 mg/ml in the application buffer) may selectively elute some proteins.
6. Regenerate the column with 2–3 bed volumes of 8 M urea, 1.5 M NaCl in PBS. Follow the urea wash with 3–5 bed volumes of application buffer. The column is now ready for re-use.
7. When not in use, store the column at 4°C with 0.02% sodium azide added as a preservative.

Application
Purification of Human Thrombospondin³

Thrombospondin was purified by a modification of the method of Lawler, et al.4 After fibrin removal, crude thrombospondin from 8 units of fresh human platelet concentrate was applied to a Bio-Gel P-300 column (6 x 45 cm) in 0.02 M Tris, 0.25 M NaCl, 2.5 mM EDTA, pH 7.4. Void volume fractions were pooled and applied (a) to the Affi-Gel heparin column (1 x 5 cm). The Affi-Gel heparin column was washed with the same buffer until the O.D.₂₈₀ returned to baseline (b). NaCl concentration was established, initially, by eluting with a NaCl gradient (0.15 to 2.0 M NaCl in Tris, EDTA buffer). Data courtesy of M.J. Doyle and D.F. Mosher, Department of Medicine, University of Wisconsin.

References
Farooqui, A. A., J. Chromatography, 184, 335 (1980).
Jaques, L. B., Pharmacological Reviews, 31, 99 (1980).
Doyle, M. J. and Mosher, D. F., Dept. of Medicine, University of Wisconsin. We thank M. Doyle and D. Mosher for their assistance in the evaluation of this product.
Lawler, J. W., Slayter, H. S. and Coligan, J. E., J. Biol. Chem., 253, 8609 (1978).

* In order of increasing retention by support.

The Affi-Prep polymyxin support was developed to meet the requirements of both the research and the process scale laboratory. Because of its unique polymer structure, the Affi-Prep polymyxin support can be sanitized by treatment with 0.1 N sodium hydroxide without affecting its ability to bind endotoxins. The support binds endotox in molecules with high capacity and selectivity, and displays the high rigidity needed in scale-up applications. Furthermore, Affi-Prep polymyxin beads are macroporous, to allow efficient surface area utilization and high throughput.

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