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10011164A.qxp 11/19/2007 9:54 AM Page II Table of Contents Section 1 ...Introduction ........................................1 Section 2 Product Information............................2 Section 3 Connection to Low-Pressure Chromatography Systems ..................7 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr. Hercules, CA 94547 USA 510-741-1000 1-800-424-6723 Section 4 Connection to Medium- and HighPressure Chromatography Systems .11 4.1 BioLogic DuoFlow™ Systems .................................
10011164A.qxp 11/19/2007 9:54 AM Page IV Section 9 Scaling Up........................................23 Section 10 Regenerating, Cleaning, and Storage .....................................23 Section 11 Troubleshooting Guide......................26 Section 12 Ordering Information.........................28 Section 13 References .......................................29 Section 14 Legal Notices ...................................
10011164A.qxp 11/19/2007 9:54 AM Page 2 Section 2 Product Information Bio-Scale Mini cartridges are prepacked chromatography cartridges supplied ready for use in 1 ml and 5 ml sizes. Bio-Scale Mini cartridges can be used with any liquid chromatography system capable of setting a high pressure limit of 45 psi (equivalent to 3 bar or 300 kPa). Alternatively, luer fittings offer convenient connection directly to a Luer-Lok syringe for quick, one-step purification.
10011164A.qxp 11/19/2007 9:54 AM Page 4 Table 2. Profinity eXact resin specifications. Functional ligand Mutant subtilisin Table 3. Buffer and chemical compatibilities for Profinity eXact cartridges. Base bead Superflow 6% agarose Particle size range 60–160 µm Reagent Group Recommended linear flow rate <1,000 cm/hr at 25°C Triggering Ions Description F-, Cl-, N3-, NO2-, CO2- Salts Sodium Acetate £3M NaCl or KCl Do not use in lysis and wash buffers.
10011164A.qxp 11/19/2007 9:54 AM Page 6 Reagent Group Description Stability Denaturants Guanidine-HCl Do not use in lysis and wash buffers. Urea Up to 2 M with no drop in binding capacity. At 4 M there may be some loss of binding capacity. At 8 M, binding capacity and target protein purity will be reduced. CaCl2 £5 mM when used with MES, MOPS, or PIPES buffers. Do not use with phosphate buffers. MgCl2 £5 mM. Do not use with fluoride containing buffers. Use NaN3 as an alternate triggering ion.
10011164A.qxp 11/19/2007 9:54 AM Page 8 Platen pressure screw SAMPLE LOOP SAMPLE LOOP INJECT PORT INJECT PORT Lock-ring Tubing Luer fittimg 1 4 7 m Alar C 2 5 8 0 3 6 9 WASTE FILL TO BIOLOGIC LP SYSTEM OR ECONO GRADIENT PUMP MV-6 INJECT VALVE TO COLUMN TO COLUMN "FILL" POSITION "INJECT" POSITION WASTE . Fig. 1. BioLogic LP system setup. (Use orange lock rings and medium size barb fittings with 1.6 mm tubing.) 2.
10011164A.qxp 4. 11/19/2007 9:54 AM Page 10 Connect the cartridge outlet to the 1.6 mm ID tubing leading to the BioLogic LP system optics module or to the Model EM-1 Econo UV monitor. It is recommended to use the shortest length (approximately 10 cm) of 1.6 mm ID tubing. Connect a barb to female luer to the 1.6 mm ID tubing, then connect to the bottom of the male luer on the Bio-Scale Mini cartridge.
10011164A.qxp 11/19/2007 9:54 AM Page 12 4.3 FPLC Systems Fig. 4. Luer to 1/4-28 adaptor. 4.2 HPLC Systems The luer to 10-32 adaptor fittings kit (catalog #732-0112) provides fittings necessary to connect the Bio-Scale Mini cartridge to nut and ferrule type fittings found on most HPLC systems. Alternatively, the cartridge can be connected to HPLC systems via a low dead-volume 1/16 inch union with a new piece of stainless-steel tubing attached to the union. Simply slip a short length of the 0.
011164A.qxp 11/19/2007 9:54 AM Page 14 Section 5 Buffers and Methods Table 4. Buffer composition. Bio-Scale Mini Profinity eXact cartridges can be run using either native or denaturing purification protocols. Under native conditions, proteins are purified using buffers that help retain the natural folded structure of the target protein. Under denaturing conditions, a strong chaotrope (e.g.
10011164A.qxp 11/19/2007 9:54 AM Page 16 Section 6 Quick Solubility Screening Protocol Before choosing a native or denaturing purification protocol, it is useful to determine the approximate expression level of a protein, and to determine if the overexpressed target protein partitions into the soluble or insoluble fraction. Soluble proteins are typically purified with the native purification procedure, while insoluble proteins can be solubilized in urea and purified with the denaturing procedure.
10011164A.qxp 11/19/2007 9:54 AM Page 18 Section 7 Preparation of E. coli Lysate For E. coli cultures expressing medium to high levels of Profinity eXact-tagged proteins, (≥10% of total protein), 50 ml of culture will yield sufficient material for a 1 ml cartridge purification, and 250 ml of culture will yield sufficient material for a 5 ml cartridge purification run.
10011164A.qxp 5. 11/19/2007 9:54 AM Page 20 Remove the supernatant, and filter through a 0.45 µM filter before applying to the cartridge. Section 8 Cartridge Preparation and Purification Protocol To prepare a cartridge, remove the end plugs and connect the cartridge to the chromatography system. The cartridge is now ready for use. The following 1 ml cartridge purification protocol is a general guideline for first time users. Flow rates for 5 ml cartridges are shown in parentheses.
10011164A.qxp 11/19/2007 9:54 AM kD Tube #: 1 2 3 4 567 9 2.00 1112 Page 22 1 Lane Order 2 3 4 5 250 150 1.75 100 1.50 75 1.25 50 37 1.00 0.75 Cleavage Incubation 0.50 25 20 0.25 0.00 15 10.00 AU 20.00 30.00 40.00 10 Min.Tenth Fig. 6. Native purification. Purification of Profinity eXact-tagged Maltose Binding Protein from the soluble fraction using a BioLogic DuoFlow system. 5 ml of lysate (5 CV) prepared from 50 ml of E. coli culture was loaded onto a 1 ml cartridge.
10011164A.qxp 11/19/2007 9:54 AM Page 24 cartridge after five purifications, however, the following regeneration procedure may be used to prolong the useful lifespan of a cartridge. In addition, in order to avoid cross-contamination it is recommended that single cartridges be designated for single proteins. In order to reuse a cartridge, the Profinity eXact tag, which is bound tightly to the functional ligand, MUST be removed.
10011164A.qxp 11/19/2007 9:54 AM Page 26 Section 11 Troubleshooting Guide Problem Possible Cause Solution Cartridge clogging or slow flow rate Particulates in samples or buffers Filter all samples and buffers through 0.45 µM filter prior to application Sample too viscous Add nuclease to lysate to degrade DNA Low level of target protein in starting material Check expression level by SDS-PAGE Target protein not binding Used resin must be regenerated before reuse.
10011164A.qxp 11/19/2007 9:54 AM Page 28 Section 12 Ordering Information Section 13 References Cartridges Ruan, B et al. Engineering subtilisin into a fluoridetriggered processing protease useful for one-step purification.
10011164A.qxp 11/19/2007 9:54 AM Page 30 Profinity eXact™ vectors, tags, and resins are exclusively licensed under patent rights of Potomac Affinity Proteins. This product is intended for research purposes only. For commercial applications or manufacturing using these products, commercial licenses can be obtained by contacting the Life Science Group Chromatography Marketing Manager, Bio-Rad Laboratories, Inc., 6000 Alfred Nobel Drive, Hercules, CA 94547, Tel (800) 4BIORAD.