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10006400B.qxp 7/10/2006 3:48 PM Page III Table of Contents Section 1 ...Introduction ........................................1 Section 2 Connecting to Bio-Rad’s Low-Pressure Chromatography Instruments ........................................5 Section 3 Connection to Other Liquid Chromatography Systems ................10 3.1 BioLogic DuoFlow™ Systems.................................10 3.2 HPLC Systems ......................11 3.3 FPLC Systems .......................
10006400B.qxp 7/10/2006 3:48 PM Page IV 5.1 Regeneration .........................18 5.2 Cleaning.................................18 5.3 Storage..................................20 Section 6 Technical Assistance ........................20 Section 7 Ordering Information.........................21 Section 8 References .......................................
10006400B.qxp 7/10/2006 3:48 PM Page 1 Section 1 Introduction Bio-Scale Mini cartridges have a patent-pending double-wall design that provides extra durability and allows easy, reliable runs with aqueous buffers commonly used for protein purification. The polypropylene luer fittings and internal sealing surfaces ensure leak-free operation at pressures up to 45 psi. Bio-Scale Mini cartridges are convenient, disposable, and supplied ready for use.
10006400B.qxp 7/10/2006 3:48 PM Page 2 Bio-Scale Mini Macro-Prep High Q and S and Macro-Prep DEAE cartridges are packed with Macro-Prep® ion exchange media. These media are based on hydrophilic spherical polymeric beads designed for the purification of proteins, nucleic acids, viruses, plasmids, and other macromolecules. Macro-Prep beads are designed to provide medium capacity, low backpressure, and high productivity. Detailed product information is given in Tables 1 and 2.
10006400B.qxp 7/10/2006 3:48 PM Page 3 Table 1. Bio-Scale Mini Macro-Prep High Q, High S, and DEAE cartridge specifications. Sizes Dimensions 1 ml and 5 ml bed volumes 1 ml: 40 mm length x 5.6 mm inner diameter 5 ml: 40 mm length x 12.
10006400B.qxp 7/10/2006 3:48 PM Page 4 Table 2. Macro-Prep High Q & S and Macro-Prep DEAE specifications.
10006400B.qxp 7/10/2006 3:48 PM Page 5 Section 2 Connecting to Bio-Rad’s Low-Pressure Chromatography Instruments Bio-Scale Mini cartridges are ideal for use with Bio-Rad's BioLogic™ LP chromatography system, Econo™ gradient pump, the patented* Model EP-1 Econo™ pump, and all low-pressure chromatography instruments. Bio-Scale Mini cartridges can be conveniently connected directly to the system using the luer fittings on the cartridge. 1. Install 1.6 mm inner diameter (ID) tubing in the pumphead.
006400B.qxp 7/10/2006 3:48 PM Page 6 Assemble with fittings and lock rings as shown in Figure 1. (Use orange lock rings and medium size barb fittings with 1.6 mm tubing.) Platen pressure Platen pressure screw screw Lock-ring Lock-ring Tubing Tubing Luer Luerfittimg fitting See detail See detail 1 4 7 rm Ala C 2 5 8 0 3 6 9 . Fig. 1. BioLogic LP system setup.
10006400B.qxp 2. 7/10/2006 3:48 PM Page 7 To maximize gradient accuracy and to 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 now correspond to the printed diagram on the valve (see Figure 2).
10006400B.qxp 7/10/2006 3:48 PM Page 8 3. Connect the inlet of the cartridge to the male luer fitting on the MV-6 sample inject valve (see 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. For optimum performance, a cartridge should be mounted vertically with the arrow on the cartridge pointing downward (see Figure 3). 4. Connect the cartridge outlet to the 1.
10006400B.qxp 7/10/2006 3:48 PM Fig. 3. Cartridge and fittings.
10006400B.qxp 7/10/2006 3:48 PM Page 10 Section 3 Connecting to Other Liquid Chromatography Systems 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 bar are usually caused by restrictions in tubing or detector cells downstream from the cartridge.
10006400B.qxp 7/10/2006 3:48 PM Page 11 Fig. 4. Luer to 1/4-28 adaptor. 3.2 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.
10006400B.qxp 7/10/2006 3:48 PM Page 12 3.3 FPLC Systems The luer to M6 adaptor fittings kit (catalog #732-0111) provides fittings necessary to connect the Bio-Scale Mini cartridge to the M6 fittings found on FPLC or related systems. Alternatively, connection can be made by using one GE Healthcare Union luer lock female to M6 female fitting (GE 18-1027-12) and one female luer lock to M6 male fitting (Upchurch P-686 or GE 18-1027-62).
10006400B.qxp 7/10/2006 3:48 PM Page 13 Section 4 Preparing a Cartridge For Use Bio-Scale Mini Macro-Prep High Q, Macro-Prep High S, and Macro-Prep DEAE cartridges contain 20% ethanol (v/v) as the storage solution. The fully hydrated support is ready to use after equilibrating the cartridge in the buffer of choice. To perform buffer exchange, connect the cartridge to a liquid chromatography system or peristaltic pump and condition it as instructed below: 1. Set pump flow rate to 3.
10006400B.qxp 7/10/2006 3:48 PM Page 14 4. Equilibrate the cartridge with low-salt buffer for 5 min. 5. Reduce the flow rate to that which will be used in the purification protocol. 4.1 Sample Preparation Proper pH and ionic strength are necessary for consistent and reproducible results. Sample can be exchanged into the starting buffer or diluted to the starting buffer's concentration.
10006400B.qxp 7/10/2006 3:48 PM Page 15 Table 3. Products for buffer exchange. Sample Volume Recommended Product Use Catalog # 50–100 µl Bio-Spin 6 column Desalting proteins ≥ 6 kD 732-6002 50–100 µl Bio-Spin 30 column Desalting proteins ≥ 30 kD 732-6006 100 µl–3 ml Bio-Scale Mini P-6 cartridge Desalting proteins ≥ 6 kD 732-4502 Up to 3 ml Econo-Pac 10DG desalting column Desalting proteins ≥ 6 kD 732-2010 Unlimited Bio-Gel P-6DG gel Desalting proteins ≥ 6 kD 150-0738 4.
10006400B.qxp 7/10/2006 3:48 PM Page 16 gradient from 0.0 to 0.4 M NaCl spanning 1 to 20 column volumes at 120 cm/hr, or 0.5 ml/min for the 1 ml cartridge and 2.5 ml/min for the 5 ml cartridge. The separation can be optimized by changing the gradient profile. At the end of each run, the cartridge can be regenerated with 1.0 M NaCl followed by starting buffer. Return to the desired flow rate and proceed with the next separation. 4.
10006400B.qxp 7/10/2006 3:48 PM Page 17 Table 4. Common buffers for ion exchange chromatography. Type of Buffering Buffer Range Cation Exchanger Acetic acid Citric acid HEPES Lactic acid MES MOPS Phosphate PIPES Pivalic acid TES Tricine Anion Exchanger Bicine Bis-Tris Diethanolamine Diethylamine L-Histidine Imidazole Pyridine Tricine Triethanolamine Tris 4.8–5.2 4.2–5.2 7.6–8.2 3.6–4.3 5.5–6.7 6.5–7.9 6.7–7.6 6.1–7.5 4.7–5.4 7.2–7.8 7.8–8.9 7.6–9.0 5.8–7.2 8.4–8.8 9.5–11.5 5.5–6.0 6.6–7.1 4.9–5.6 7.
10006400B.qxp 7/10/2006 3:48 PM Page 18 Section 5 Care of the Cartridge 5.1 Regeneration After each use, the cartridge should be regenerated with the appropriate salt, in most cases 1–2 M NaCl in the presence of buffer. Wash with 2 to 4 column volumes of the buffered high salt solution. This reduces the potential for protein precipitation when selecting acid as a cleaning agent. 5.
10006400B.qxp 7/10/2006 3:48 PM Page 19 appropriate cleaning protocol. As a general guide, we recommend the following: 1. Use high salt buffer for regeneration, as above. 2. For aggregated or precipitated proteins, or when dirty feed stock (e.g., crude lysate) has been used, wash with 3–5 column volumes of 6 M guanidine-HCl or 8 M urea at 100 cm/hr. 3. For lipids or hydrophobically bound contaminants, wash with 0.1% Triton X-100 or 20–70% ethanol or isopropyl alcohol, or 1–30% acetic acid.
10006400B.qxp 7/10/2006 3:48 PM Page 20 is equilibrated in the starting buffer before loading the sample. 5.3 Storage After washing the cartridges with deionized water, Bio-Scale Mini ion exchange cartridges should be purged and stored with PBS, containing 0.05% NaN3, or in 20% (v/v) ethanol solution, and capped for extended storage.
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10006400B.qxp 7/10/2006 3:48 PM Page 23 Section 8 References 1. Gagnon P, Avoiding Instrument-associated Aberrations in Purification Scale-up and Scaledown, BioPharm 10, 42–45 (1997) 2. Harris ELV and Angal S, Protein Purification Methods: A Practical Approach, IRL Press, Oxford (1989) 3. Scopes RK, Protein Purification: Principles and Practice (Second Edition), Springer-Verlag, New York (1987) 4.
10006400B.qxp 7/10/2006 3:48 PM Page II Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
