Purification of DNA-Binding Protein Using HiTrap Heparin HP

Characteristics of Heparin Sepharose and Capto Heparin chromatography media</p>

	Ligand density (mg/ml)	Compositionp	pH stability1	Average particle size (µm)
Heparin Sepharose High Performance	10	Heparin coupled to Sepharose High Performance by reductive amination to give a stable attachment even in alkaline conditions.	Short term: 5 to 10 Long term: 5 to 10	34
Heparin Sepharose 6 Fast Flow	5.0	Heparin coupled to Sepharose 6 Fast Flow by reductive amination to give a stable attachment even in alkaline conditions.	Short term: 4 to 13 Long term: 4 to 12	90
Capto Heparin	1.8	Heparin coupled to Capto	Short term: 4 to 13 Long term: 4 to 12	90

1 Short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance.

HiTrap Heparin HP and  Heparin Sepharose 6 Fast Flow

Heparin Binding buffer: 20 mM Tris-HCl, pH 8.0 or 10 mM sodium phosphate, pH 7.0

Heparin Elution buffer: 20 mM Tris-HCl, 1 to 2 M NaCl, pH 8.0 or 10 mM sodium phosphate, 1 to 2 M NaCl, pH 7.0

Equilibrate the column with 10 CV of binding buffer.

Load the sample.

Wash with 5 to 10 CV of Heparin binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm).

Elute with 5 to 10 CV of Heparin elution buffer using a continuous or step gradient from 0% to 100% elution buffer.

Example

After Purification from Ni, Desalt using buffer:  20 mM Tris-HCl, pH 8, 5% (v/v) glycerol,  10 mM 2-mercaptoethanol,   0.1 M NaCl

Then load 35 ml  to  HiTrap Heparin HP 5 ml column.

Binding buffer: 20 mM Tris-HCl, pH 8, 5% (v/v) glycerol, 10 mM 2-mercaptoethanol, 0.1 M NaCl

Elution buffer: 20 mM Tris-HCl, pH 8, 5% (v/v) glycerol,  10 mM 2-mercaptoethanol,   1 M NaCl

Flow rate: 1 ml/min (30 cm/h)

Gradient: Linear, 0.1 M NaCl to 1 M NaCl in 20 column volumes

Fraction volume: 3 ml

Cleaning

Remove ionically bound proteins by washing with 0.5 CV of 2 M NaCl for 10 to 15 min.

Remove precipitated or denatured proteins by washing with 4 CV of 100 mM NaOH for 1 to 2 h; or 2 CV of 6 M guanidine hydrochloride for 30 to 60 min; or 2 CV of 8 M urea for 30 to 60 min. Remove hydrophobically bound proteins by washing with 4 CV of 0.1% to 0.5% Tween 20 for 1 to 2 h.

Chemical stability

100 mM NaOH (1 w at 20°C), 50 mM sodium acetate, pH 4.0, 4 M NaCl, 8 M urea, 6 M guanidine hydrochloride.

Storage

Wash chromatography media and columns with 50 mM sodium acetate containing 20% ethanol (use approximately 5 CV for packed media) and store at 4°C to 8°C.

Capto Heparin

Binding buffer: 100 mM Tris-HCl, 10 mM trisodium citrate, 225 mM NaCl, pH 7.4

Wash buffer: 100 mM Tris-HCl, 10 mM trisodium citrate, 330 mM NaCl, pH 7.4

Elution buffer: 100 mM Tris-HCl, 10 mM trisodium citrate, 2 M NaCl, pH 7.4

Equilibrate with 5 CV of binding buffer.

Apply the sample.

Wash step 1: wash with 40 CV of binding buffer.

Wash step 2: wash with 15 CV of wash buffer.

Elute with 9.5 CV of elution buffer.

A flow rate of 0.5 ml/min is recommended for a 1 ml column.

Cleaning

Substances such as denatured proteins that do not elute during regeneration can be removed by cleaning-in-place (CIP) procedures.

A recommended CIP procedure for Capto Heparin is 4 CV of 100 mM NaOH with a contact time of 1 to 2 h.

Storage

Store unused chromatography media at 4ºC to 30ºC in 20% ethanol and 50 mM of sodium acetate.