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        亲和层析凝胶指南

        互联网

        3421

        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 of Union Carbide 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 capacitundefined: 13 mg trypsin/ml drained media
        Bead structure: 6% agarose
        Bead size range: 45�165 μm
        Mean particle size: 90 μm
        Max linear flow ratundefined~I_75_cm~Hh_at_25°C, HR 16/10 column,
        5 cm bed height
        pH stabilitundefined*~Kbr_~H~M~2~1~0Long_term~I_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
        ~undefined The binding capacity was determinded in 50 mM Tris-HCI,
        pH 8.0 containing 0.5 M NaCl.
        ~undefined_Linear_flow_rate_~L~Kbr_~H~M~2~1~0Volumetric_flow_rate_~Acm3~Hh~B~Kbr_~H~M~2~1~0column_cross~Fsectional_area_~Acm2~B~Kbr_~H~M~2~1~undefined*_The_ranges_given_are_estimates_based_on_our_knowledge_and~Kbr_~H~M~2~1~0experience._Please_note_the_following~I~Kbr_~H~M~2~1~0pH_stability~E_long_term_refers_to_the_pH_interval_where_the_medium~Kbr_~H~M~2~1~0is_stable_over_a_long_period_of_time_without_adverse_effects_on_its~Kbr_~H~M~2~1~0subsequent_chromatographic_performance.~Kbr_~H~M~2~1~0pH_stability~E_short_term_refers_to_the_pH_interval_for_regeneration~Kbr_~H~M~2~1~0and_cleaning.Contents~Kbr_~H~M~2~1~01._Preparing_the_medium_3~Kbr_~H~M~2~1~02._Packing_Sepharose_6B_medium_4~Kbr_~H~M~2~1~03._Using_an_adaptor_5~Kbr_~H~M~2~1~04._Binding_of_protein_5~Kbr_~H~M~2~1~05._Elution_of_protein_6~Kbr_~H~M~2~1~06._Regeneration_6~Kbr_~H~M~2~1~07._Cleaning_7~Kbr_~H~M~2~1~08._Storage_7~Kbr_~H~M~2~1~09._Ordering_Information_7~Kbr_~H~M~2~1~010._References_7~Kbr_~H~M~2~1~01._Preparing_the_medium~Kbr_~H~M~2~1~0Benzamidine_Sepharose_6B_is_supplied_pre~Fswollen_in_20~7~Kbr_~H~M~2~1~0ethanol._Prepare_a_slurry_by_decanting_the_ethanol_solution~Kbr_~H~M~2~1~0and_replacing_it_with_binding_buffer_in_a_ratio_of_75~7_settled~Kbr_~H~M~2~1~0media_to_25~7_buffer_before_packing._The_binding_buffer~Kbr_~H~M~2~1~0should_not_contain_agents_which_significantly_increase_the~Kbr_~H~M~2~1~0viscosity._The_column_may_be_equilibrated_with_viscous~Kbr_~H~M~2~1~0buffers_at_reduced_flow_rates_after_packing_is_completed.2._Packing_Sepharose_6B_medium~Kbr_~H~M~2~1~01._Equilibrate_all_material_to_the_temperature_at_which_the~Kbr_~H~M~2~1~0chromatography_will_be_performed.~Kbr_~H~M~2~1~02._De~Fgas_the_medium_slurry.~Kbr_~H~M~2~1~03._Eliminte_air_from_the_column_dead_spaces_by_flushing~Kbr_~H~M~2~1~0the_end_pieces_with_buffer._Make_sure_no_air_has_been~Kbr_~H~M~2~1~0trapped_under_the_column_net._Close_the_column_outlet~Kbr_~H~M~2~1~0with_a_few_centimeters_of_buffer_remaining_in_the~Kbr_~H~M~2~1~0column.~Kbr_~H~M~2~1~04._Pour_the_slurry_into_the_column_in_one_continuous~Kbr_~H~M~2~1~0motion._Pouring_the_slurry_down_a_glass_rod_held_against~Kbr_~H~M~2~1~0the_wall_of_the_column_will_minimize_the_introduction_of~Kbr_~H~M~2~1~0air_bubbles.~Kbr_~H~M~2~1~05._Immediately_fill_the_remainder_of_the_column_with~Kbr_~H~M~2~1~0buffer~E_mount_the_column_top_piece_onto_the_column_and~Kbr_~H~M~2~1~0connect_the_column_to_a_pump.~Kbr_~H~M~2~1~06._Open_the_bottom_outlet_of_the_column_and_set_the_pump~Kbr_~H~M~2~1~0to_run_at_the_desired_flow_rate._This_should_be_at_least~Kbr_~H~M~2~1~0133~7_of_the_flow_rate_to_be_used_during_subsequent~Kbr_~H~M~2~1~0chromatographic_procedures._However~E_the_maximum~Kbr_~H~M~2~1~0flow_rate~E_see_Table_1~E_is_typically_employed_during~Kbr_~H~M~2~1~0packing.~Kbr_~H~M~2~1~0Note~I_I_f_you_have_packed_at_the_maximum_linear_flow~Kbr_~H~M~2~1~0rate~E_do_not_exceed_75~7_of_this_in_subsequent~Kbr_~H~M~2~1~0chromatographic_procedures.~Kbr_~H~M~2~1~07._Maintain_the_packing_flow_rate_for_3_bed_volumes_after_a~Kbr_~H~M~2~1~0constant_bed_height_is_reached.~Kbr_~H~M~2~1~0For_detailed_desription_on_column_packing~E_refer_to_our~Kbr_~H~M~2~1~0Handbook_on_Affinity_Chromatography~E_Principles_and~Kbr_~H~M~2~1~0Methods_~A18~F1022~F29~B~E_which_can_be_downloaded_from~Kbr_~H~M~2~1~0~K~Hfont~M_~Kfont_color~L~4~522229c~4~Mwww.gehealthcare.com~Hprotein~Fpurification3.~K~Hfont~M__~Kfont~M_Using_an_adaptor~Kbr_~H~M~2~1~0Adaptors_should_be_fitted_as_follows~I~Kbr_~H~M~2~1~01._After_the_medium_has_been_packed_as_described_above~E~Kbr_~H~M~2~1~0close_the_column_outlet_and_remove_the_top_piece_from~Kbr_~H~M~2~1~0the_column._Carefully_fill_the_rest_of_the_column_with~Kbr_~H~M~2~1~0buffer_to_form_an_upward_meniscus_at_the_top.~Kbr_~H~M~2~1~02._Insert_the_adaptor_at_an_angle_into_the_column~E_ensuring~Kbr_~H~M~2~1~0that_no_air_is_trapped_under_the_net.~Kbr_~H~M~2~1~03._Make_all_tubing_connections_at_this_stage._There_must_be~Kbr_~H~M~2~1~0a_bubble~Ffree_liquid_connection_between_the_column_and~Kbr_~H~M~2~1~0the_bump~E_and_column_and_the_sample_application_valve.~Kbr_~H~M~2~1~04._Slide_the_plunger_slowly_down_the_column_so_that_the_air~Kbr_~H~M~2~1~0above_the_net_and_in_the_capillary_tubings_is_displaced_by~Kbr_~H~M~2~1~0eluent._Valves_on_the_inlet_side_of_the_column_should_be~Kbr_~H~M~2~1~0turned_in_all_directions_during_this_procedure_to_ensure~Kbr_~H~M~2~1~0that_air_is_removed.~Kbr_~H~M~2~1~05._Lock_the_adaptor_in_position_on_the_medium_surface~E~Kbr_~H~M~2~1~0open_the_column_outlet_and_start_the_eluent_flow._Pass~Kbr_~H~M~2~1~0eluent_through_the_column_at_the_packing_flow_rate_until~Kbr_~H~M~2~1~0the_medium_bed_is_stable._Re~Fposition_the_adaptor_on_the~Kbr_~H~M~2~1~0medium_surface_as_necessary.~Kbr_~H~M~2~1~0The_column_is_now_equilibrated_and_ready_for_use.~Kbr_~H~M~2~1~04._Binding_of_protein~Kbr_~H~M~2~1~0A_suitable_binding_buffer_is_50_mM_Tris~FHCl~E_pH_8.0_containing~Kbr_~H~M~2~1~00.5_M_NaCl._Good_results_are_obtained_at_room_temperature~Kbr_~H~M~2~1~0although_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.

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