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GE Healthcare
Instructions 71-5000-15 AD Pre-activated media
CNBr-activated
Sepharose 4 Fast Flow
Introduction
The preparation and use of affinity chromatography media by coupling biospecific
ligands to CNBr-activated matrices is a widely used, successful and well-documented
technique.
TM
CNBr-activated Sepharose 4 Fast Flow is a pre-activated affinity matrix that
combines the advantages of CNBr coupling with the high flow and stability
characteristics of Sepharose 4 Fast Flow. In our exper
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Table of contents 1. Product description 2 2. Coupling 3 3. Column packing guidelines 5 4. Evaluation of packing 7 5. Cleaning, Sanitization and Storage 10 6. Ordering information 11 1. Product description CNBr-activated Sepharose 4 Fast Flow is a bead-formed, highly cross- linked pre-activated matrix produced by reacting Sepharose 4 Fast Flow with cyanogen bromide (CNBr). This coupling makes the medium more rigid which in turn improves the pressure/flow characteristics. Proteins and ot
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Table 1. Medium characteristics. Mean particle size 90 μm Particle size range 45–165 μm Bead structure Highly cross-linked 4% agarose, spherical Linear flowrate Base matrix 150–250 cm/h, 0.1 MPa (1 bar), XK 50/60 column, bed height 25 cm Swelling factor 4–5 ml drained medium/g Coupling capacity 13–26 mg α-chymotrypsinogen/ml pH stability* long term 3–11 short term (CIP) 3–11 * Refers to stability of coupling between ligand abd base matrix. Ligands can be less stable. Sepha
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In order to retain maximum binding capacity of CNBr-activated Sepharose 4 Fast Flow prior to coupling the ligand, use cold (0–4 °C) solutions. The time interval between washing and coupling must be minimised; therefore preparations of all required solutions prior to coupling is recommended. 1. Prepare the coupling solution, i.e. dissolve the ligand to be coupled in a suitable coupling buffer, e.g. 0.1 M NaHCO pH 8.3 containing 0.5 M 3 NaCl. For good coupling efficiency avoid unnecessarily d
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5. Wash away exess ligand with at least 5 medium volumes of coupling buffer. 6. After coupling, non-reacted groups on the medium should be blocked. Transfer the medium to 0.1 M Tris-HCl buffer pH 8.0 or 1 M ethanolamine pH 8.0. Let it stand for 2 hours. 7. Wash the coupled medium using alternate low and high pH. Recommended buffers are 0.1M acetate buffer pH 3-4 containing 0.5 M NaCl and 0.1 M Tris-HCl buffer pH 8–9 containing 0.5 M NaCl. A suitable procedure could be 3x1 medium volume Tr
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Large scale columns TM • BPG variable bed, glass columns. Inner diameters from 100–450 mm, bed volumes from 2.4–131 litres; bed height max 83 cm. TM • CHROMAFLOW variable bed columns. Inner diameters from 400–600 mm. 3.2 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 outle
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7. When the bed has stabilized, close the bottom outlet and stop the pump. 8. If using a packing reservoir, disconnect the reservoir and fit the adaptor to the column. 9. With the adaptor inlet disconnected, push down the adaptor approximately 2 mm into the bed, allowing the packing solution to flush the adaptor inlet. 10. Connect the pump, open the bottom outlet and continue packing. The bed will be further compressed at this point and a space will form between the bed surface and the ada
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The calculated plate number will vary depending on the test conditions and it should therefore be used as a reference value only. It is also important that conditions and equipment are kept constant so that results are comparable. Changes in solute, solvent, eluent, sample volume, flow rate, liquid pathway, temperature, etc., will influence the results. For optimal results, the sample volume should be at max. 2.5% of the column volume and the flow velocity between 15 and 30 cm/h. If an acce
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The reduced plate height is calculated: HETP/d where d is the diameter of the bead. As a guideline, a value of <3 is normally acceptable. The peak should be symmetrical, and the asymmetry factor as close as possible to 1 (values between 0.8–1.5 are usually acceptable). A change in the shape of the peak is usually the first indication of bed deterioration due to use. Peak asymmetry factor calculation: A = b/a S where a = 1st half peak width at 10% of peak height b = 2nd half peak width at 1
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5. Cleaning, Sanitization and Storage For best performance of coupled CNBr-activated Sepharose 4 Fast Flow over a long working life, follow the general procedures described below. In all cases, we recommend testing the procedures at small scale first. Equilibration After packing, and before a chromatographic run, equilibrate with working buffer by washing with at least 5 bed volumes. Cleaning-In-Place Cleaning-in-place, (CIP), is a cleaning procedure which removes contaminants such as lipids
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or Wash the column with 3– column volumes of 70% ethanol. Wash immediately with at least 5 column volumes of sterile filtered binding buffer. Sanitization Sanitization inactivates microbial contaminants in the packed column and related equipment. A specific sanitization protocol should be designed for each process according to the type of contaminants present and stability of coupled ligand. Following are generally recommended procedures. Equilibrate with a buffer consisting of 2% hibitan
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