Chelating Sepharose Fast Flow
Instructions 71–5001–87 AE
Affinity chromatography
GE Healthcare
Immobilized metal ion affinity chromatography (IMAC) exploits a molecule’s affinity for chelated metal ions. The amino acid histidine present in many proteins forms complexes with transition metal ions such as Cu 2+, Zn 2+, Ni 2+ and Fe 3+. Chelating
Sepharose™ Fast Flow with a suitable immobilized metal ion will therefore selectively retain proteins with exposed histidine. Exposed cysteine and tryptophane residues may also be involved in the binding to an immobilized metal ion but their contribution to the binding is much lower than the contribution from exposed histidine residues.The strength of binding is affected by the buffer pH and the metal ion selected.Chelating Sepharose Fast Flow consists of iminodiacetic acid groups coupled to Sepharose 6 Fast Flow by stable ether linkages via a 7-atom spacer.Chelating Sepharose Fast Flow belongs to the BioProcess™ Media family.
BioProcess media are developed and supported for production scale
雅马哈xj600chromatography. All BioProcess media are produced with validated methods and are tested to meet manufacturing requirements. Secure ordering and delivery routines give a reliable supply of media for production scale. Regulatory Support Files (RSF) are available to assist process validation and submissions to regulatory authorities.
Tables of contents
1. Product description 3
2. Column packing 3
3. Evaluation of packing 6
4. Immobilized Metal Ion Affinity Chromatography (IMAC) 8
5. Choice of metal ions 11
6. Regeneration, Cleaning, Sanitization and Storage. 12
金属材料
天敏电视卡驱动7. Ordering information 14 p. 2 1. Product description
Table 1. Medium characteristics
Total capacity: 30–37 µmol Cu2+/ml drained
medium
Bead structure: 6% highly cross-linked agarose
Bead size range: 45–165 µm
Mean particle size: approx. 90 µm
Linear flow velocity: >300 cm/h at 25 °C, 0.1 MPa (1 bar,
14.5 psi), XK50/30 column, 15 cm bed
height
Max. operating pressure: 0.3 MPa (3 bar, 42 psi)
pH stability*
Long term: 3–13
Short term: 2–14
Chemical stability: All commonly used aqueous
buffers, 0.01 M HCl, 1.0 M NaOH,
20% ethanol (tested at 40 °C for 7 days) Physical stability: Negligible volume variation due to
changes in pH or ionic strength Autoclavable: In 0.1 M sodium acetate at 121 °C
for 30 min
* The ranges given are estimates based on our knowledge and experience. Please note the following:
pH stability, long term refers to the pH interval where the medium is stable over a long period
of time without adverse effects on its subsequent chromatographic performance.
pH stability, short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
2. Column packing
Chelating Sepharose Fast Flow is supplied pre-swollen in 20% ethanol. Prepare a slurry by decanting the 20% ethanol solution and replacing it with distilled water in a ratio of 75% settled medium to 25% distilled water.
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Table 2. Recommended lab-scale columns for Chelating Sepharose Fast Flow.
Packing flow rate (ml/min) Max. recommended flow rate) Empty Column* first step second step for chromatography (ml/min) Tricorn™ 10/20 0.9 4.7 2
Tricorn 10/50 0.9 4.7 2
Tricorn 10/100 0.9 4.7 2
XK 16/20 2.5 8.7 5
XK 26/20 6.6 23 13
XK 50/20 24.5 85 49聚乙二醇单甲醚
XK 50/30 24.5 85 49
* For inner diameter and maximum bed volumes and bed heights, see Ordering information
Table 3. Recommended process-scale columns for Chelating Sepharose Fast Flow
Column Inner diam Bed volume Bed height
(mm) (L) (max (cm) BPG™ 100/500 100 up to 2.0 L 26
BPG 140/500 140 up to 4.0 L 26
BPG 200/500 200 up to 8.2 L 26
BPG 300/500 300 up to 18.0 L 26
BPG 450/500 450 up to 36.0 L 23
Chromaflow™ 400/100-300 400 13–37 L 30
Chromaflow 600/100-300 600 28–85 L 30
dtap. 4
Packing lab-scale columns
1. Assemble the column (and packing reservoir if necessary).
2. Remove air from the end-piece and adapter by flushing with water. Make
sure no air has been trapped under the column bed support. Close the
column outlet leaving the bed support covered with water.
3. Resuspend the medium and 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.
4. If using a packing reservoir, immediately fill the remainder of the
column and reservoir with water. Mount the adapter or lid of the packing reservoir and connect the column to a pump. Avoid trapping air bubbles under the adapter or in the inlet tubing.
5. Open the bottom outlet of the column and set the pump to run at the
desired flow rate. Ideally, Sepharose 6 Fast Flow media are packed in XK or Tricorn columns in a two-step procedure: Do not exceed 0.5 bar (0.05 MPa) in the first step and 1.5 bar (0.15 MPa) in the second step.
If the packing equipment does not include a pressure gauge, use a
packing flow rate of 2.5 ml/min (XK 16/20 column) or 0.9 ml/min (Tricorn 10/100 column) in the first step, and 8.7 ml/min (XK 16/20 column) or
4.7 ml/min (Tricorn 10/100 column) in the second step. See Table 3 for
packing flow rates for other columns.
If the recommended pressure or flow rate cannot be obtained, use
the maximum flow rate your pump can deliver. This should also give a
wellpacked bed.
Note:For subsequent chromatography procedures, do not exceed 75% of the packing flow rate. See Table 3 for flow rates for
chromatography.
6. Maintain packing flow rate for at least 3 bed volumes after a constant
bed height is reached. Mark the bed height on the column.通信与信息管理
7. Stop the pump and close the column outlet.
8. If using a packing reservoir, disconnect the reservoir and fit the adapter
to the column.
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