Heparin-Sepharose CL-2B preparation experiment

Summary

This experiment describes the preparation of heparin-Sepharose CL-2B. This experiment was derived from the Experimental Guide for Protein Purification and Identification by Houzhu Zhu.

Operation method

Heparin-Sepharose CL-2B preparation experiment

Materials and Instruments

SepharoseCL-2B Acetonitrile Cyanogen bromide Glycine NaOH Heparin Na2CO3 NaHCO3 Ethanolamine hydrochloride NaAc NaCl Urea NaCl3 Tris-HCl EDTA Sodium azide

Move

makings

SepharoseCL-2B (PharmaciaBiotech, Inc.)

Acetonitrile

Cyanogen bromide (CNBr)(AldrichChemicalCo.C9149-2)

Glycine

NaOH

Heparin (sodium salt; USP grade; from porcine intestinal mucus, 140 U/mg) [LifeTechnologies, Inc.(GIBCO/BRL) 15077-027] (500,000U required per preparation)

Reagents

_Na2CO3 (2mol/L)

_NaHCO3 (0.1 ml/L; PH 9.5)

_NaHCO3 (0.2 mol/L; pH 8.5)

Ethanolamine hydrochloride (1 mol/L; pH 8.0)

NaAc (0.1mol/L;pH4.0)/NaCl 0.5mol/L)

Urea (2mol/L)/NaCl (0.5mol/L)

_NaHCO3 (0.lmol/L; pH 10.0) _/NaCl3 (0.5 ml/L)

Tris-HCl (100 mmol/L; pH 7.9)/EDTA (2 mmol/L)/sodium azide (0,04%, w/v)

(For the formula, see "Preparation of reagents", pp.131~138)

Operating procedure

This protocol is based on a modification of the March et al. (1974) method.

1) Wash 250 ml SepharoseCL-2B (in bed volume) with at least 1000 ml of redistilled water.
Note: Handle SepharoseCL-2B gently to avoid damaging the agarose gel particles.

2) In a 2000-ml plastic beaker, mix 250 ml of washed SepharoseCL-2B with 750 ml of _2mol/LNa2CO3 and stir with a magnetic stirrer.

3) In a fume hood, add 12.5 ml of acetonitrile to 25 g of cyanogen bromide and dissolve with glass beech stirring.

4) Transfer the solution of stibium bromide to the stirred SepharoseCL-2B at room temperature using a pasteurized pipette.

5) Stir the mixture for 2 min at room temperature.

6) Transfer the mixture to a 600-ml coarse sintered glass funnel and quickly and gently wash the mixture with 1000 ml of 0.1 _mol/LNaHCO3 (pH 9.5), 1000 ml of redistilled water and 1000 ml of 0.2 mol/LNaHC3 (pH 8.5).
Note: Do not allow the cyanogen bromide activated gel to dry to a cake!
Add 20 g glycerol and 20 g NaOH to the filtrate and leave overnight in a fume hood before discarding.

7) Dissolve 3 g of heparin (500,000 U) in 250 ml of 0.2 mol/L _NaHCO3 (PH 8.5) in a 500-ml plastic bottle.
Note: Heparin products purchased from different manufacturers have very different properties when used in heparin-agarose chromatography. In fact, some heparin products are completely ineffective for heparin-agarose chromatography, but of course we are not obliged to use heparin products from a specific source (GIBCO/BRL) as listed in the "Materials" section.

8) Transfer the cyanogen bromide-activated gel to a 500-ml plastic vial containing heparin solution (all solutions should be at room temperature).

9) Mix for 20 h at 4°C.

10) Collect the heparin-sepharose gel by filtration through a sintered glass funnel. Suspend with 400 ml of 1moI/L ethanolamine hydrochloride (pH 8.0) and mix for at least 4 h at room temperature.

11) The gel was washed with 2000 ml of 0.lmol/LNaOAc (pH 4.0)/0.5mol/LNaCl, 2000 ml of 2mol/L urea/0.5mol/LNaCl, 2000 ml of 0. _1mol/LNaHCO3 (pH 10.0)/0.5mol/LNaCl, and 2000 ml of re-distilled water, respectively.

12) Suspend the prepared gel in 250 ml of 100 mmol/LTris-HCl (pH 7.9)/2 mmol/LEDTA/0.04% sodium azide.

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