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10011433A.qxp 10/30/2007 8:21 AM Page 2 Table of Contents Section 1 ...Introduction. .......................................1 Section 2 ...Connecting to Bio-Rad’s Low..............Pressure Chromatography ..............Instruments ........................................5 Section 3 ..Connecting to Other Liquid ..............Chromatography Systems ................10 ..............3.1 3.2 3.3 BioLogic DuoFlow™ Systems.................................11 HPLC Systems ......................11 FPLC Systems ..
10011433A.qxp 10/30/2007 5.2 5.3 5.4 8:21 AM Page 4 Wash Alternatives ..................20 Autoclaving ............................20 Storage..................................20 Section 6 ..Technical Support.............................21 Section 7 ..Ordering Information.........................22 Section 8 ..References .......................................
10011433A.qxp 10/30/2007 8:21 AM Page 5 Section 1 Introduction Bio-Scale™ Mini cartridges are easy-to-use, prepacked chromatographic cartridges for fast, reproducible chromatographic separations. They have a double-wall design that provides extra durability and allows easy, reliable runs with aqueous buffers most commonly used for protein purification. The polypropylene luer fittings and internal sealing surfaces assure leak-free operation, at pressures up to 45 psi.
10011433A.qxp 10/30/2007 8:21 AM Page 2 The Bio-Scale Mini CHT cartridges are packed with Type I or Type II ceramic hydroxyapatite supports. These supports are based on hydroxyapatite, a form of calcium phosphate used in chromatographic separations of biomolecules. CHT ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite. Unlike most other chromotagraphic absorbents, CHT is both the ligand and the support matrix.
10011433A.qxp 10/30/2007 8:21 AM Page 4 Table 2. Product Description Functional groups Type I Type II Ca2+, PO4, OH Ca2+, PO4, OH ≥ 25 mg Lys/g CHT ≥ 12.5 mg Lys/g CHT Observed dynamic binding capacity lysozyme (Lys) Nominal pore diameter 600–800 Å 800–1,000 Å Maximum backpressure 100 bar (1,500 psi) 100 bar (1,500 psi) Nominal mean particle size Bulk density 20 ± 2, 40 ± 4, and 80 ± 8 µm 0.63 g/ml 0.
10011433A.qxp 10/30/2007 8:21 AM Page 6 (Use orange lock rings and medium size barb fittings with 1.6 mm tubing.) Platen pressure screw Lock-ring Tubing 2. To maximize gradient accuracy and apply samples efficiently, install 1.6 mm ID tubing from the pump to the MV-6 sample inject valve (if available). If using the MV-6 sample inject valve, turn the knob counterclockwise as far as it will go so it will correspond to the printed diagram on the valve. (Figure 2).
10011433A.qxp 10/30/2007 8:21 AM Page 8 3. Hold the cartridge vertically with outlet in the downward direction. Connect the inlet of the cartridge to the male luer fitting on the MV-6 sample inject valve (Figure 2). If not using the MV-6 sample inject valve, connect a barb to male luer fitting on the 1.6 mm ID tubing, then connect to the top of the female luer on the Bio-Scale mini cartridge.
10011433A.qxp 10/30/2007 8:21 AM Page 10 Section 3 Connecting to Other Liquid Chromatography Systems The Bio-Scale Mini cartridges can be connected to any liquid chromatography system, provided that the maximum pressure limit (3 bar, 45 psi, or 300 KPa) of the cartridges is not exceeded. It is recommended that the system pressure limit be set according to the cartridge pressure limit. Pressures in excess of 3.
011433A.qxp 10/30/2007 8:21 AM Page 12 3.3 FPLC Systems The luer to M6 adaptor fittings kit (catalog number 732-0111) provides fittings necessary to connect the cartridge to the M6 fittings found on FPLC or related systems. Alternatively, connection can be made by using the following Upchurch Scientific® Quick Connect Luer Adaptors: two Upchurch P-621 adaptor, 1/4–28, to metric adaptors, one Upchurch P-619 adaptor, 1/4–28, to male luer, and one Upchurch P-628 adaptor, 1/4–28, to female luer.
10011433A.qxp 10/30/2007 8:21 AM Page 14 5. Equilibrate the cartridge with running buffer and operatate the cartridge according to the CHT instruction manual or according to your protocol. Table 3. Products for Buffer Exchange Sample Volume Recommended Product 4.1 Sample Preparation 50–100 µl Bio-Spin 6 column 50–100 µl Proper pH and ionic strength are necessary for consistent and reproducible results.
10011433A.qxp 10/30/2007 8:21 AM Page 16 calcium. Common buffers for hydroxyapatite chromatography are listed in Table 4. An appropriate starting point for purifying samples is a linear gradient from 5–500 mM sodium phosphate pH 6.8, spanning 10 to 20 column volumes at 2.0 ml/min for the 5 ml cartridge. For separations of monoclonal antibodies from aggregates, an alternative purification protocol is eluting with a linear gradient from 0-2 M sodium chloride in 5-20 mM sodium phosphate, pH 6.5.
10011433A.qxp 10/30/2007 8:21 AM Page 18 CHT Suitability pH Buffer 10 cycles 7.0 50 cycles MES + 5 mM PO4 + n/a 7.0 Acetate + 5 mM PO4 + n/a 7.0 Imidazole + 5 mM PO4 + + 7.0 Glycine + 5 mM PO4 + n/a 7.0 Arginine + 5 mM PO4 + n/a 7.0 HEPES + 5 mM PO4 + n/a 7.0 Tris + 5 mM PO4 + n/a 7.5 Phosphate (5 mM) + n/a 7.5 MES + 5 mM PO4 + n/a 7.5 Imidazole + 5 mM PO4 + + 7.5 Acetate + 5 mM PO4 + n/a 7.5 HEPES + 2 mM PO4 + n/a 7.5 HEPES + 5 mM PO4 + n/a 7.
10011433A.qxp 10/30/2007 8:21 AM Page 20 5.2 Wash Alternatives Perform wash alternatives with any of the following alternative buffers in place of step 1. Continue with step 2 listed in Section 5.1. • 1–2 M KCI or NaCI • 6 M urea • 8 M guanidine-HCI All the wash alternative buffers should contain 5 mM phosphate at neutral pH. 5.
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10011433A.qxp 10/30/2007 8:21 AM Page 24 Section 8 References M. Gorbunoff et al., The Interaction of Proteins with Hydroxyapatite II: Role of Acidic and Basic Groups, Analytical Biochemistry 136, 433–439 (1984) P. Gagnon et al., A Ceramic Hydroxyapatite-Based Purification Platform, BioProcess International 4 (2), 50–60 (2006) M. Gorbunoff et al., The Interaction of Proteins with Hydroxyapatite III: Mechanism., Analytical Biochemistry 136, 440–445 (1984) T. Ogawa et al.
10011433A.qxp 10/30/2007 8:21 AM Page 26 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
