GE HealthcareBenzamidine Sepharose™ 6B is p-aminobenzamidine covalentlyattached to Sepharose 6B by the epoxy coupling method.p-Aminobenzamidine (PAB), is a synthetic inhibitor of trypsin-likeserine protease. Trypsin and trypsin-like serine proteases bind toBenzamidine 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 serineproteases that have been purified on Benzamidine Sepharose 6B.For recombinant purification, Benzamidine Sepharose 6B can be usedfor removal of serine proteases such as thrombin and enterokinaseafter cleavage of purification tags.Sample TextSepharose and Drop Design are trademarks of GE Healthcare companies. GE, imagination at work and GE monogram aretrademarks 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 whichsupplies them. GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to makechanges in specifications and features shown herein, or discontinue the product described at any time without notice orobligation. 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 GmbHMunzinger Strasse 5D-79111 FreiburgGermanyGE Healthcare UK Ltd.Amersham PlaceLittle ChalfontBuckinghamshire, HP7 9NAUKGE Healthcare Bio-Sciences Corp.800 Centennial AvenueP.O. Box 1327Piscataway, NJ 08855-1327USAGE Healthcare Bio-Sciences KKSanken Bldg.3-25-1, HyakuninchoShinjuku-ku, Tokyo 169-0073Japanwww.gehealthcare.com/protein-purificationwww.gehealthcare.comGE Healthcare Bio-Sciences ABBjörkgatan 30751 84 UppsalaSweden71-7096-00 AE 07/2006Elanders Östervåla 2006 12345Elanders Östervåla 2006 12345Elanders Östervåla 2006Elanders Östervåla 2006Elanders Östervåla 2006 12345Elanders Östervåla 2006 12345Elanders Östervåla 2006Elanders Östervåla 2006imagination at workTable 1. Medium characteristics.Ligand density: 7 μmole p-aminobenzamidine/mldrained mediaAvailable capacity*: 13 mg trypsin/ml drained mediaBead structure: 6% agaroseBead size range: 45–165 μmMean particle size: 90 μmMax linear flow rate**: 75 cm/h at 25°C, HR 16/10 column,5 cm bed heightpH stability***Long term: 3–11Short term: 2–13Chemical stability: Stable to all commonly used aqueousbuffersPhysical stability: Negligible volume variation due tochanges 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 andexperience. Please note the following:pH stability, long term refers to the pH interval where the mediumis stable over a long period of time without adverse effects on itssubsequent chromatographic performance.pH stability, short term refers to the pH interval for regenerationand cleaning.Contents1. Preparing the medium 32. Packing Sepharose 6B medium 43. Using an adaptor 54. Binding of protein 55. Elution of protein 66. Regeneration 67. Cleaning 78. Storage 79. Ordering Information 710. References 71. Preparing the mediumBenzamidine Sepharose 6B is supplied pre-swollen in 20%ethanol. Prepare a slurry by decanting the ethanol solutionand replacing it with binding buffer in a ratio of 75% settledmedia to 25% buffer before packing. The binding buffershould not contain agents which significantly increase theviscosity. The column may be equilibrated with viscousbuffers at reduced flow rates after packing is completed.2. Packing Sepharose 6B medium1. Equilibrate all material to the temperature at which thechromatography will be performed.2. De-gas the medium slurry.3. Eliminte air from the column dead spaces by flushingthe end pieces with buffer. Make sure no air has beentrapped under the column net. Close the column outletwith a few centimeters of buffer remaining in thecolumn.4. Pour the slurry into the column in one continuousmotion. Pouring the slurry down a glass rod held againstthe wall of the column will minimize the introduction ofair bubbles.5. Immediately fill the remainder of the column withbuffer, mount the column top piece onto the column andconnect the column to a pump.6. Open the bottom outlet of the column and set the pumpto run at the desired flow rate. This should be at least133% of the flow rate to be used during subsequentchromatographic procedures. However, the maximumflow rate, see Table 1, is typically employed duringpacking.Note: I f you have packed at the maximum linear flowrate, do not exceed 75% of this in subsequentchromatographic procedures.7. Maintain the packing flow rate for 3 bed volumes after aconstant bed height is reached.For detailed desription on column packing, refer to ourHandbook on Affinity Chromatography, Principles andMethods (18-1022-29), which can be downloaded fromwww.gehealthcare.com/protein-purification3. Using an adaptorAdaptors should be fitted as follows:1. After the medium has been packed as described above,close the column outlet and remove the top piece fromthe column. Carefully fill the rest of the column withbuffer to form an upward meniscus at the top.2. Insert the adaptor at an angle into the column, ensuringthat no air is trapped under the net.3. Make all tubing connections at this stage. There must bea bubble-free liquid connection between the column andthe bump, and column and the sample application valve.4. Slide the plunger slowly down the column so that the airabove the net and in the capillary tubings is displaced byeluent. Valves on the inlet side of the column should beturned in all directions during this procedure to ensurethat air is removed.5. Lock the adaptor in position on the medium surface,open the column outlet and start the eluent flow. Passeluent through the column at the packing flow rate untilthe medium bed is stable. Re-position the adaptor on themedium surface as necessary.The column is now equilibrated and ready for use.4. Binding of proteinA suitable binding buffer is 50 mM Tris-HCl, pH 8.0 containing0.5 M NaCl. Good results are obtained at room temperaturealthough the optimal temperature for binding is 4°C.After the sample has been loaded, wash the medium withbinding buffer until the baseline is stable.5. Elution of proteinBound substances can be eluted specifically or nonspecifically.To elute bound substances specifically, acompeting 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 ofbound sucstances:• A change in ionic strenght alters the degree of ionizationof the charged groups at the binding site. Elution isnormally complete at salt concentrations of 1 M or less ofNaCl. Either step or continuous gradients may be used.• A change in pH alters the degree of ionization of thecharged groups at the binding. Either step or continuousgradients may be used.• Reduction of the polarity of the elution buffer byadditionof 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 guanidinehydrochloride is an alternative for elution of boundsubstances.6. RegenerationDepending of the nature of the sample, BenzamidineSepharose 6B may be regenerated for re-use by washingthe 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 sodiumacetate + 0.5 M NaCl, pH 4.5) buffers. This cycle should berepeated 3 times followed by re-equilibration with 3–5 bedvolumes of binding buffer.If detergent or denaturing agents have been used during chromatography,these can also be used in the washing buffer.7. CleaningIn some applications, substances like denaturated proteinsor lipids do not elute in the regeneration procedure. Thesecan be removed by washing the column with a detergentsolution, e.g. 0.1% Triton X-100 at 37°C for one minute.Re-equilibrate immediately with at least 5 bed volumes ofbinding buffer.8. StorageBenzamidine Sepharose 6B should be stored at 4–8°C inthe presence of a suitable bacteriostatic agent, e.g. 20%ethanol, at neutral pH.9. Ordering InformationProduct Pack size Code No.Benzamidine Sepharose 6B 25 ml 17-0568-0110. References1. Purification and characterization of a trypsin-like serineproteinase from rat brain slices that degrades lamininand type IV collagen and stimulates protease-activatedreceptor-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 affinitychromatography 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 Complexwith Peptide and MHC Class II. Science 1999 December 3;286: 1913–1921. Reinherz, Ellis L. et al.