heparin sepharose 6FF 说明书

GE HealthcareInstructions 71-7190-00 AE Affinity media

Heparin Sepharose 6 Fast FlowHeparin is a naturally occurring glycosaminoglycan which serves as an effective affinity binding and ion exchange ligand for a wide range of biomolecules, including coagulation factors and other plasma proteins, lipoproteins, protein synthesis factors, enzymes that act on nucleic acids and steroid receptors. By coupling heparin to TM Sepharose 6 Fast Flow with a chemically optimized linkage, GE Healthcare provides an excellent medium for both laboratory and process scale affinity purification.

Contents

1. Medium characteristics

2. Packing columns

3. Evaluation of packing

4. Operation

5. Maintenance

6. Ordering information

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1. Medium characteristicsHeparin is a glycosaminoglycan consisting of alternating hexuronic (D-glucuronic or L-iduronic) and D-glucosamine residues. The polymer is heavily sulphated, carrying sulphamino (N-sulphate) groups at C-2 of the glucosamine units as well as ester sulphate (O-sulphate) groups in various positions. (Figure 1). The heparin ligand used in Heparin Sepharose 6 Fast Flow is isolated from porcine intestinal mucosa, and has a molecular weight distribution over the range 5 000–30 000. The base matrix, Sepharose 6 Fast Flow, consists of highly cross-linked 6% agarose beads. It provides the medium with high physical stability and excellent flow characteristics. Heparin is linked to the Sepharose matrix by reductive amination which is a very stable binding, even in alkaline conditions. The stability of this medium is limited only by the heparin itself. The essential characteristics of Heparin Sepharose 6 Fast Flow are summarized in Table 1.(A) (B)

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Figure 1. Heparin consists of alternating hexuronic acid (A) and D-glucosamine residues (B). The hexuronic acid can be either D-glucuronic acid (top) or its C-5 epimer, L-iduronic acid. R1=–H or–SO3; R2=–SO3 or–COCH3.

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Table 1. Characteristics of Heparin Sepharose 6 Fast Flow.

Ligand density approx 4 mg heparin/ml drained mediumAverage particle size 90 μm (45–165 μm)

Bead structure 6% highly cross-linked spherical agarose,Flow rate ≥300 cm/h at 100 kPa (XK 50/60 column, bed height 25-cm, eluent distilled water, 20 °C)Recommended pH

working and long term 4–12

short term, (cleaning in place) 4–13

Chemical stability (1 week, 40 °C) 0.01 M NaOH

0.05 M sodium acetate, pH 4.0

4 M NaCl

8 M urea

6 M guanidine hydrochloride

(1 week, 20 °C) 0.1 M NaOH

Autoclavable 121 °C for 20 minutes in distilled water

Delivery and storage conditions Supplied in 0.05 M sodium acetate containing 20% ethanol

2. Packing columns

Heparin Sepharose 6 Fast Flow is supplied pre-swollen. Decant the 20%

ethanol solution and replace it with binding buffer before use.

Recommended columns

Lab-scale columns

Tricorn 5/20 (5 mm i.d.) for bed volumes up to 0.55 ml at bed heights up

to 2.8 cm

Tricorn 5/50 (5 mm i.d.) for bed volumes up to 1.14 ml at bed heights up

to 5.8 cm

Tricorn 10/20 (10 mm i.d.) for bed volumes up to 2.2 ml at bed heights up

to 2.8 cm

Tricorn 10/50 (10 mm i.d.) for bed volumes up to 4.56 ml at bed heights up

to 5.8 cm

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Tricorn 10/100 (10 mm i.d.) for bed volumes up to 8.48 ml at bed heights

up to 10.8 cm

XK 16/20 (16 mm i.d.) for bed volumes up to 30 ml at bed heights up to

15 cm.

XK 26/20 (26 mm i.d.) for bed volumes up to 80 ml at bed heights up to

15 cm.

XK 50/20 (50 mm i.d.) for bed volumes up to 275 ml at bed heights up to

15 cm.

Large scale columns

BPG variable bed, glass columns. Inner diameters from 100–450 mm,

bed volumes from 2.4–131 litres; bed height max 83 cm.

BioProcess Stainless Steel (BPSS) fixed bed columns. Inner diameters

from 400–1400 mm; bed volumes from 12–1500 litres, bed height

10–100 cm.

INdEX variable bed columns. Inner diameters from 70–200 mm; bed

volumes up to 24.8 litres; bed heights of max 79 cm.

CHROMAFLOW variable bed columns. Inner diameters from

280–2000 mm.TMTMTMTM

Packing lab-scale columns

1. Assemble the column (and packing reservoir if necessary).

2. Remove air from the column dead spaces by flushing the end-piece

and adaptor with packing buffer. Make sure no air has been trapped

under the column bed support. Close the column outlet leaving the bed

support covered with packing buffer.

3. Resuspend medium stored in its container by shaking (avoid stirring the

sedimented medium). Mix the packing buffer with the medium to form

50–70% slurry (sedimented bed volume/slurry volume = 0.5–0.7).

4. Pour the slurry into the column in a single continuous motion. Pouring

the slurry down a glass rod held against the column wall will minimize

the introduction of air bubbles.

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5. If using a packing reservoir, immediately fill the remainder of the

column and reservoir with packing buffer. Mount the adaptor or lid of

the packing reservoir and connect the column to a pump. Avoid

trapping air bubbles under the adaptor or in the inlet tubing.

6. Open the bottom outlet of the column and set the pump to run at the

desired flow rate. Ideally, Sepharose 6 Fast Flow based media are

packed at a constant pressure of approximately 1.5 bar (0.15 MPa). If the packing equipment does not include a pressure gauge, use a packing

flow velocity of approximately 500 cm/h (10 cm bed height, 25 °C, low

viscosity buffer).

If the recommended pressure or flow rate cannot be obtained, use

the maximum flow rate the pump can deliver. This s …… 此处隐藏：19188字，全部文档内容请下载后查看。喜欢就下载吧 ……