亲和层析凝胶指南

GE Healthcare Benzamidine Sepharose™ 6B is p-aminobenzamidine covalently attached to Sepharose 6B by the epoxy coupling method. p-Aminobenzamidine (PAB), is a synthetic inhibitor of trypsin-like serine protease. Trypsin and trypsin-like serine proteases bind to Benzamidine Sepharose 6B and can thus be used for purification and/ or removal of these substances. Trypsin, bovine thrombin, urokinase, human enterokinase, acrosin, native plasminogen, kallikrein, prekallikrein, collagenase and clostripain are some of the serine proteases that have been purified on Benzamidine Sepharose 6B. For recombinant purification, Benzamidine Sepharose 6B can be used for removal of serine proteases such as thrombin and enterokinase after cleavage of purification tags. Sample TextSepharose and ｄｒｏｐ Design are trademarks of GE Healthcare companies. GE, imagination at work and GE monogram are trademarks of General Electric Company. Triton is a trademark ofｕｎｉｏｎCarbide Chemicals and Plastics Co. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your local GE Healthcare representative for the most current information. © 2006 General Electric Company � All rights reserved. GE Healthcare AB, a General Electric Company. GE Healthcare Europe GmbH Munzinger Strasse 5 D-79111 Freiburg Germany GE Healthcare UK Ltd. Amersham Place Little Chalfont Buckinghamshire, HP7 9NA UK GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ 08855-1327 USA GE Healthcare Bio-Sciences KK Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073 Japan www.gehealthcare.com/protein-purification www.gehealthcare.com GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala Sweden 71-7096-00 AE 07/2006 Elanders Östervåla 2006 12345 Elanders Östervåla 2006 12345 Elanders Östervåla 2006 Elanders Östervåla 2006 Elanders Östervåla 2006 12345 Elanders Östervåla 2006 12345 Elanders Östervåla 2006 Elanders Östervåla 2006 imagination at workTable 1. Medium characteristics. Ligand density: 7 μmole p-aminobenzamidine/ml drained media Available capacity*: 13 mg trypsin/ml drained media Bead structure: 6% agarose Bead size range: 45�165 μm Mean particle size: 90 μm Max linear flow rate**: 75 cm/h at 25°C, HR 16/10 column, 5 cm bed height pH stability*** Long term: 3�11 Short term: 2�13 Chemical stability: Stable to all commonly used aqueous buffers Physical stability: Negligible volume variation due to changes in pH or ionic strength * The binding capacity was determinded in 50 mM Tris-HCI, pH 8.0 containing 0.5 M NaCl. ** Linear flow rate = Volumetric flow rate (cm3/h) column cross-sectional area (cm2) *** 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 and cleaning.Contents 1. Preparing the medium 3 2. Packing Sepharose 6B medium 4 3. Using an adaptor 5 4. Binding of protein 5 5. Elution of protein 6 6. Regeneration 6 7. Cleaning 7 8. Storage 7 9. Ordering Information 7 10. References 7 1. Preparing the medium Benzamidine Sepharose 6B is supplied pre-swollen in 20% ethanol. Prepare a slurry by decanting the ethanol solution and replacing it with binding buffer in a ratio of 75% settled media to 25% buffer before packing. The binding buffer should not contain agents which significantly increase the viscosity. The column may be equilibrated with viscous buffers at reduced flow rates after packing is completed.2. Packing Sepharose 6B medium 1. Equilibrate all material to the temperature at which the chromatography will be performed. 2. De-gas the medium slurry. 3. Eliminte air from the column dead spaces by flushing the end pieces with buffer. Make sure no air has been trapped under the column net. Close the column outlet with a few centimeters of buffer remaining in the column. 4. Pour the slurry into the column in one continuous motion. Pouring the slurry down a glass rod held against the wall of the column will minimize the introduction of air bubbles. 5. Immediately fill the remainder of the column with buffer, mount the column top piece onto the column and connect the column to a pump. 6. Open the bottom outlet of the column and set the pump to run at the desired flow rate. This should be at least 133% of the flow rate to be used during subsequent chromatographic procedures. However, the maximum flow rate, see Table 1, is typically employed during packing. Note: I f you have packed at the maximum linear flow rate, do not exceed 75% of this in subsequent chromatographic procedures. 7. Maintain the packing flow rate for 3 bed volumes after a constant bed height is reached. For detailed desription on column packing, refer to our Handbook on Affinity Chromatography, Principles and Methods (18-1022-29), which can be downloaded from www.gehealthcare.com/protein-purification3. Using an adaptor Adaptors should be fitted as follows: 1. After the medium has been packed as described above, close the column outlet and remove the top piece from the column. Carefully fill the rest of the column with buffer to form an upward meniscus at the top. 2. ｉｎｓｅｒｔ the adaptor at an angle into the column, ensuring that no air is trapped under the net. 3. Make all tubing connections at this stage. There must be a bubble-free liquid connection between the column and the bump, and column and the sample application valve. 4. Slide the plunger slowly down the column so that the air above the net and in the capillary tubings is displaced by eluent. Valves on the inlet side of the column should be turned in all directions during this procedure to ensure that air is removed. 5. Lock the adaptor in position on the medium surface, open the column outlet and start the eluent flow. Pass eluent through the column at the packing flow rate until the medium bed is stable. Re-position the adaptor on the medium surface as necessary. The column is now equilibrated and ready for use. 4. Binding of protein A suitable binding buffer is 50 mM Tris-HCl, pH 8.0 containing 0.5 M NaCl. Good results are obtained at room temperature although the optimal temperature for binding is 4°C. After the sample has been loaded, wash the medium with binding buffer until the baseline is stable.5. Elution of protein Bound substances can be eluted specifically or nonspecifically. To elute bound substances specifically, a competing agent such as p-aminobenzamidine can be used. Competitive elution buffer: 20 mM p-aminobenzamidine in binding buffer. Several methods may be used for non-specific elution of bound sucstances: • A change in ionic strenght alters the degree of ionization of the charged groups at the binding site. Elution is normally complete at salt concentrations of 1 M or less of NaCl. Either step or continuous gradients may be used. • A change in pH alters the degree of ionization of the charged groups at the binding. Either step or continuous gradients may be used. • Reduction of the polarity of the elution buffer byaddition of dioxane (up to 10%) or ethylene glycol (up to 50%) may be used for elution of bound substances. • Use of deforming agents like urea or guanidine hydrochloride is an alternative for elution of bound substances. 6. Regeneration Depending of the nature of the sample, Benzamidine Sepharose 6B may be regenerated for re-use by washing the medium with 2�3 bed volumes of alternating high pH (0.1 M Tris-HCl + 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate + 0.5 M NaCl, pH 4.5) buffers. This cycle should be repeated 3 times followed by re-equilibration with 3�5 bed volumes of binding buffer. If detergent or denaturing agents have been used during chromatography, these can also be used in the washing buffer.7. Cleaning In some applications, substances like denaturated proteins or lipids do not elute in the regeneration procedure. These can be removed by washing the column with a detergent solution, e.g. 0.1% Triton X-100 at 37°C for one minute. Re-equilibrate immediately with at least 5 bed volumes of binding buffer. 8. Storage Benzamidine Sepharose 6B should be stored at 4�8°C in the presence of a suitable bacteriostatic agent, e.g. 20% ethanol, at neutral pH. 9. Ordering Information Product Pack size Code No. Benzamidine Sepharose 6B 25 ml 17-0568-01 10. References 1. Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease-activated receptor-2. J. Neurochem. (2000), 74(4), 1731�1738, Sawada, K. et al. 2. Purification of mast cell proteases from murine skin. Exp. Dermatol. (1999), 8(5), 413�418, Algermissen, B. et al. 3. Purification of rabbit liver aldehyde oxidase by affinity chromatography on benzamidine Sepharose 6B. J. Chromatogr. (1989), 475 363-72, Stell, J. G. P. et al. 4. The Crystal Structure of a T Cell Receptor in Complex with Peptide and MHC Class II. Science 1999 December 3; 286: 1913�1921. Reinherz, Ellis L. et al.