Heparin-agarose

Preparation of Heparin-agarose

1. Wash 500 ml settled bed volume of Biogel A 1.5M (Biorad, 151-0450)  twice with distilled water by decantation and suspended in 1.5 litres of distilled water.

2. Dissolve 25 g of CNBr (C91492-25G, $48) in 50 ml of cold dimethylformamide.

3. Add to the agarose suspension with stirring in an ice bath.

4. maintain the pH of the mixture between 10.5 and 11.5 with 5 N NaOH and let the reaction continue until the rate of change of the pH with time become negligible (about one hour).

5. Pour the suspension to a column.

6. Wash activated agarose with 5 1 of distilled water.

All of these operations were carried out in a fume cupboard.

7. Suspend  agarose cake in 1 1 of 0.1 M NaHCO3 containing 1 g of heparin (Sigma).

8. Stir the suspension slowly overnight at 4 oC.

9. Add 50 ml of triethylamine

10. Stire for 4 hours.

11. Pour the agarose to a column.

12. Wash with 2 1 of distilled water.

13. Suspend the agarose in an equal volume of buffer containing 20 mM Tris-HCl pH 7.5 and 0.5 mM EDTA.

14. Store agarose at 4°. Stable and active for more than two years

Protocol 2

1. Wash a 250 ml volume of settled bed Sepharose resin with 2 liters of ddH2O in a 600 ml scintered glass funnel using a 2 liter side arm flask under a modest vacuum. Do NOT let the bed resin run dry.

2. Transfer the resin to a 2 liter glass beaker using a large spatula and resuspend the resin in 750 ml 4°C ddH2O. Add a large magnetic stir bar to the beaker and place it into an ice bucket so that ice surrounds the sides of the beaker. It is convenient to cover the beaker with plastic wrap using a rubber band to secure the plastic wrap to prevent ice getting into the resin.

3. CAUTION!! (see Hint #1) To resuspend 25 g of cyanogen bromide in 50 ml dimethyl formamide (DMF), first add a small stir bar and about 10 to 15 ml DMF to the bottle of cyanogen bromide and then start the solution stirring on a stir plate in a fume hood. Then transfer the cyanogen bromide liquid to a beaker and rinse the bottle with fresh DMF and pool into a beaker with stirring.

4. Add the cyanogen bromide liquid to the Sepharose (on ice in a fume hood) slowly with stirring using a disposable 10 ml glass pipet. Using 5 M NaOH, keep the pH of the reaction between pH 10.5 and 11.5 for 1 hr. This will require about 100 ml 5 M NaOH per 250 ml Sepharose. Check the pH with pH strips.

5. Wash the cyanogen bromide-activated Sepharose with 4 liters of 4°C ddH2O on a 600 ml scintered glass funnel under a modest vacuum. Do NOT let the bed resin run dry. Dispose of filtrate and first wash in appropriate waste.

6. Transfer the cyanogen bromide-activated Sepharose with a large spatula to 750 ml 4°C 0.1 M NaHCO3 containing 3 g (500,000 Units) of the sodium salt of heparin (see Hint #2).

7. Stir slowly overnight (12 to 18 hr) at 4°C (in a cold room).

8. Add 30 ml triethylamine and stir for 4 hr at 4°C.

9. Wash the resin on a 600 ml scintered glass funnel with 1.5 liters of ddH2O

10. Wash the resin on the scintered glass funnel with 1.5 liters of TEN.

11. Transfer the resin to a 500 ml wide mouth polypropylene bottle and fill it with TEN Azide. Store at 4°C.

Buffer

0.1 M Sodium Bicarbonate

0.1 M Sodium Bicarbonate (NaHCO3)

TEN Azide 0.02% (w/v)

Sodium Azide 0.02% (w/v)

Sodium Azide (NaN3, CAUTION! see Hint #1)
1 mM EDTA
300 mM NaCl
10 mM Tris-HCl, pH 7.5

TEN

0.3 M NaCl
1 mM EDTA
10 mM Tris-HCl, pH 7.5

5 M NaOH

Protocol 3, Couple heparin to  CyBr activated Sepharose 4B

Heparin (Vetren, 125 IU/mg, USP XVII)

Heparin-agarose chromatography of DNA/RNA binding proteins (restriction enzyme, ligase etc)

1. Package a column with 20-25 ml heparin-agarose resin.

25 ml resin is good for purifying restriction enzymes from  100 g cell pellet.

2. After purification on phosphocellulose column, collect and pool fractions containing enzymatic activity. Dialyse agaist Buffer H (low salt buffer) o/n.

3. Load to Heparin-agarose column. Wash with low salt buffer.

4. Elute with 10 CV linear gradient of NaCl, 0~1 M