Editing Purification of DNA-Binding Protein Using HiTrap Heparin HP

Characteristics of Heparin Sepharose and Capto Heparin chromatography media&lt;/p&gt;

{|
|-
! <br/>
! Ligand density (mg/ml)
! width="20%" | Compositionp
! pH stability1
| style="vertical-align: top;" | 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.
| width="20%" | Short term: 5 to 10<br/>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.
| width="20%" | Short term: 4 to 13<br/>Long term: 4 to 12
| 90
|-
| Capto Heparin
| 1.8
| Heparin coupled to Capto
| width="20%" | Short term: 4 to 13<br/>Long term: 4 to 12
| 90
|}

1&nbsp;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&nbsp;&nbsp;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:&nbsp;&nbsp;20 mM Tris-HCl, pH 8, 5% (v/v) glycerol,&nbsp; 10 mM 2-mercaptoethanol,&nbsp; &nbsp;0.1 M NaCl

Then load&nbsp;35 ml&nbsp; to&nbsp; 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,&nbsp; 10 mM 2-mercaptoethanol,&nbsp; &nbsp;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.
[[Category:Protein Purification]]