Operating Instructions
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1 POROS Affinity Depletion Cartridges Operating Instructions 1 Product Description POROS Affinity Depletion Cartridges and columns are available in two affinity chemistries: POROS Anti-HSA Cartridges Packed with POROS Perfusion Chromatography media covalently coupled with an affinity-purified goat polyclonal antibody against human serum albumin (HSA). POROS Protein G Cartridges Packed with POROS Perfusion Chromatography media covalently coupled with recombinant Protein G. POROS Affinity Depletion Cartridges can be operated: Singly or in tandem with a cartridge of a different chemistry. Manually or on an automated MDLC (multi-dimensional liquid chromatography) workstation (for example, Applied Biosystems VISION or BioCAD workstations). Table 1 Support Matrix Product Characteristics Cross-linked poly(styrene-divinylbenzene) Immobilized Ligand POROS Anti-HSA Affinity-purified goat anti-hsa polyclonal antibody POROS G Recombinant protein G Saturation 500 cm/hr POROS Anti-HSA 2 mg/ml. For complete removal of HSA in serum samples, do not exceed 50% of the saturation capacity. For more information, see the Sample Volume ranges listed on page 3. POROS G 15 mg/ml. For complete removal of IgG, do not exceed 50% of the saturation capacity. When used in tandem with a POROS Anti-HSA cartridge, do not exceed the Anti-HSA cartridge capacity. Shipping Solvent 0.1 M phosphate buffer ph 7.0, 0.05% sodium azide Shrinkage/Swelling Cartridge Dimensions <1% from 1 to 100% solvent 4 mmd 15 mml (0.2 ml) 6.2 mmd 66.5 mml (2.0 ml) 7.5mmD 100 mml (4.4 ml) Table 2 Chemical Resistance ph Range POROS Anti-HSA 2 to 10 POROS G 2 to 9 Ionic Strength Range 0 to 5 M, all common salts Buffer Additives POROS Anti-HSA Agents that may degrade the protein ligands are not recommended. POROS G All common agents, including 4 M urea, 2 M guanidine hydrochloride, ethylene glycol, and detergents. Agents that may degrade the protein ligands are not recommended. Solvents Operating Temperature Contents Water, 0 to 30% alcohols, acetonitrile, other common organic solvents. NOTE: Do not expose to strong oxidizers (such as hypochlorite), oxidizing acids (such as nitric acid), or strong reducing agents (such as sulfite). 5 to 40 C Page 1 Product Description Connecting the Cartridge Buffers Preparing the Anti-HSA Cartridge Cartridge Operating Conditions Running a Cleanup Method Storing the Cartridge Accessories, Spare Parts, and Ordering Information Technical Support
2 2 Connecting the Cartridge POROS Affinity Depletion Cartridges can be used singly or in tandem with a second cartridge of different chemistry. 2.1 Single-Cartridge Connection Figure 1 illustrates connections of a single cartridge for manual use. For use with an automated MDLC workstation (for example, Applied Biosystems VISION or BioCAD workstations), plumb the cartridge inlet using standard 10/32 HPLC fittings (PEEK or stainless steel). 3 Buffers In general, when you prepare buffers: Use buffers of the highest purity practical. Degas and filter (0.22 or 0.45 µm) all buffers before use. Prepare the following buffers. Buffer Composition Equilibration, Load, and Wash Elution Phosphate buffered saline (PBS), ph mm HCl Syringe needle Syringe Needle-port adapter Cartridge holder with cartridge DANGER CHEMICAL HAZARD. Hydrochloric acid (HCl) causes severe eye, skin, and respiratory tract burns. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Cleaning Anti-HSA 1 M NaCl Protein G 2 M urea in 1 M NaCl or 10% acetic acid in 1 M NaCl CAUTION CHEMICAL HAZARD. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Urea may cause eye, skin and respiratory tract irritation. 10% acetic acid in 1 M sodium chloride is a flammable liquid and vapor. Exposure causes eye, skin, and respiratory tract burns. It may be harmful if inhaled, swallowed, or absorbed through the skin. Keep away from sparks, heat, and flame. Compression screw Figure 1 Cartridge Connection for Manual Use 2.2 Tandem-Cartridge Connection 1/16-inch O.D. PEEK outlet tubing For tandem-cartridge connection, connect a 5-cm piece of blue PEEK tubing (1/16-inch O.D., inch I.D.) between cartridges using standard 10/32 HPLC fittings. 4 Preparing the Anti-HSA Cartridge Before injecting human serum for the first time on the Anti-HSA cartridge, perform one blank run (no sample) that includes the following steps: 5 cartridge-volume equilibration step 5 cartridge-volume elution step 5 cartridge-volume clean step 5 cartridge-volume equilibration step The Equilibration step followed by the Elution step removes any weakly bound antibody from the cartridge and increases the run-to-run reproducibility. 2
3 5 Cartridge Operating Conditions BIOHAZARD. Biological samples such as tissues, body fluids, and blood of humans and other animals have the potential to transmit infectious diseases. Read and follow the guidelines published in Biosafety in Microbiological and Biomedical Laboratories ( and in the Occupational Safety and Health Standards, Bloodborne pathogens ( waisidx_01/ 29cfr1910a_01.htm). Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective eyewear, clothing, and gloves. Step/Buffer Condition Settings 0.2-mL Cartridge 2.0-mL Cartridge 4.4 ml-cartridge Equilibration (5 to 10 cartridge volumes) Load Anti-HSA Note: The concentration of albumin in diluted human serum is 3 to 7 mg/ml. Adjust sample volume to accommodate the albumin concentration in your sample. For example, if the albumin concentration in your 10% diluted human serum sample is ~3 mg/ml, you can load up to the maximum of 70 µl. If the albumin concentration in your sample is ~7 mg/ml, load a lower sample volume of 30 µl. Sample Volume (10% diluted human serum with PBS) 30 to 70 µl 300 to 700 µl 600 to 1,600 µl 0.5 ml/min 1.2 ml/min 1.8 ml/min Capture the Load step effluent in up to five 0.5-CV fractions. Determine which fractions contain protein (measure by UV absorbance at 280 nm), then pool the protein-containing fractions for further proteomics analysis. If breakthrough of albumin occurs, it may be because the concentration of albumin in the sample exceeds the cartridge capacity. Repeat the run and use a smaller injection volume. Load Protein G Note: The concentration of IgG in diluted human serum is 0.8 to 1.8 mg/ml. Adjust sample volume to accommodate the IgG concentration in your sample. For example, if the IgG concentration in your 10% diluted human serum sample is ~0.8 mg/ml, you can load up to the maximum of 2 ml. If the IgG concentration in your sample is ~1.8 mg/ml, load a lower sample volume of 1 ml. Sample Volume (10% diluted human serum with PBS) 1 to 2 ml 20 to 10 ml N/A 0.5 ml/min 1.2 ml/min N/A Wash Elution 12 mm HCl Cleaning Anti-HSA 1 M NaCl Protein G 2 M urea in 1 M NaCl or 10% acetic acid in 1 M NaCl (15 to 30 cartridge volumes) (5 to 10 cartridge volumes) (5 to 10 cartridge volumes) Capture the Load step effluent in up to five 0.5-CV fractions. Determine which fractions contain protein (measure by UV absorbance at 280 nm), then pool the protein-containing fractions for further proteomics analysis. For optimum performance, run an Elution step during each cycle to remove the bound serum proteins and prepare the cartridge for the next cycle. Perform a Cleaning step after 1 to 2 cycles to reduce fouling that may decrease cartridge performance. 3
4 6 Running a Cleanup Method In some applications, or after many cycles, sample molecules may not fully elute or may precipitate on the cartridge and cause any of the following symptoms: Increased bandspreading Loss of binding capacity Loss of recovery Increased pressure drop Trace or ghost peaks different from the usual RI (refractive index) shift peak during blank runs Clean the cartridge if any of the symptoms listed above appear. NOTE: In any cleanup method, reversing the flow direction is recommended to help flush out particulates and to prevent contamination of the lower part of the bed. Also, to expose the cartridge to the regeneration solution for several minutes at each step of the cleaning protocol, slowing the flow rate is recommended. Recommended Cleaning Solutions DANGER CHEMICAL HAZARD. 20% Acetic acid is a flammable liquid and vapor. Exposure causes eye, skin, and respiratory tract burns. It may be harmful if inhaled, swallowed, or absorbed through the skin. Keep away from sparks, heat, and flame. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. CHEMICAL HAZARD. Guanidine hydrochloride may be harmful if swallowed or absorbed through the skin. Exposure may cause eye, skin, and respiratory tract irritation and adverse effects on the central nervous system and bone marrow. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Cleaning by Making Multiple Injections For very aggressive or very viscous solvents, use multiple injections of cleaning solutions instead of pumping cleaning solutions directly. To clean by injections: Make the injection volume as large as possible. Use a low flow rate to expose the cartridge to the cleaning solution for several minutes. Cartridge Anti-HSA Protein G Cleaning Solution 1 M NaCl 2 M urea in 1 M NaCl 10% acetic acid in 1 M NaCl CAUTION CHEMICAL HAZARD. Urea may cause eye, skin and respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. DANGER CHEMICAL HAZARD. 10% acetic acid in 1 M sodium chloride is a flammable liquid and vapor. Exposure causes eye, skin, and respiratory tract burns. It may be harmful if inhaled, swallowed, or absorbed through the skin. Keep away from sparks, heat, and flame. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Cleaning by Pumping To clean the cartridge, follow these guidelines: 1. Wash with 15 to 30 column volumes of the solution recommended for your cartridge to remove contaminants. 2. For Protein G cartridges only, you can remove precipitated protein, bound lipids, and so on, by other means, for example: Brief washes with alcohols, acetonitrile, or mixtures of these solvents with 20% acetic acid 2 M guanidine hydrochloride CHEMICAL HAZARD. Acetonitrile (ACN) is a flammable liquid and vapor. Exposure may cause eye, skin, and respiratory tract irritation, central nervous system depression, and damage to the heart, blood system, liver, and kidneys. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 4
5 7 Storing the Cartridge When you store your cartridge, always be sure to: Store cartridges in the refrigerator. DO NOT FREEZE CARTRIDGES Store the cartridge with the endcaps in place, carefully sealed to prevent drying. Drying results in decreased chromatographic efficiency. Short-Term Storage (less than 1 day) 1. Equilibrate the cartridge in PBS. 2. Remove the cartridge from the cartridge holder and insert end caps to prevent the cartridge from drying out. 3. Store cartridges at 4 C. Long-Term Storage (more than 1 day) 1. Equilibrate the cartridge in PBS with 0.05% sodium azide. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. If disposing of solutions that contain sodium azide down laboratory sinks, flush the drains with large volumes of water to prevent azide buildup. 2. Remove the cartridge from the cartridge holder and insert end caps to prevent the cartridge from drying out. 3. Store at 4 C. 8 Accessories, Spare Parts, and Ordering Information Available accessories for your POROS Affinity Depletion cartridges are listed below: Table 3 Description POROS Affinity Depletion Anti-HSA Cartridge Affinity Cartridge Accessories Quantity Part Number 4 mmd 15 mml (0.2 ml) mmd 66.5 mml (2.0 ml) mmD 100 mml (4.4 ml) POROS Affinity Depletion Protein G Cartridge 4 mmd 15 mml (0.2 ml) mmd 66.5 mml (2.0 ml) Cartridge Holder 0.2 ml ml Needle-Port Adapter Outlet Connector Technical Support Applied Biosystems is dedicated to helping you use POROS Affinity Depletion Cartridges to the fullest extent possible. Applied Biosystems publishes a continuing series of Proteomics Application and Technical Notes. Please contact Applied Biosystems directly for a publication list. For further details or for answers to questions on POROS Affinity Depletion cartridges, Perfusion Chromatography, or other products, please contact Applied Biosystems. Refer to the back page of this document for contact information. 5
6 Copyright 2002, 2011 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. Applied Biosystems, BioCAD, Perfusion Chromatography, and POROS, are registered trademarks of Applied Biosystems or its subsidiaries in the U.S. and certain other countries. AB (Design), Applera, and VISION are trademarks of Applied Biosystems or its subsidiaries in the U.S. and certain other countries. Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone: Toll Free (In North America): Fax: Worldwide Sales Offices Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For international office locations, please call our headquarters or see our Web site at Technical Support In North America, call Outside North America, see our Web site at or call our local sales office. PEEK is a trademark of Victrex, PLC. All other trademarks are the sole property of their respective owners. Applied Biosystems is committed to providing the world s leading technology and information for life scientists. Printed in the USA, 04/2011 Part Number Rev. B
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