proteus 210 G.qxd 5/9/05 1:42 PM Page 1 GPROTEUS Protein G Antibody Purification Handbook Mini & Midi spin columns proteus 210 G.qxd 5/9/05 1:42 PM Page 1 SECTION 1 SECTION 1 SECTION 3 Introduction 2 Easy-to-read protocols for purifying 24 Proteus benefits 3 antibodies using Protein G Contents of a Proteus spin column kit 4 Delipidation procedure 25 Additional recommended equipment 8 Buffers in the kit 25 Storage conditions 8 Step by step Mini protocol 26 Technical support 9 Step by step Midi protocol 30 Specifications 10 APPENDIX SECTION 2 Procedure to convert RPM to RCF in a 35 Principles of Protein A and G chromatography 11 swing bucket rotor Affinity chromatography 12 Procedure to convert RPM to RCF in a 36 General conditions for optimal binding 16 fixed angle rotor Choosing the correct buffer conditions 17 Protein G kit buffer formulation 37 Eluting the protein from a Proteus spin column 18 Questions and answers 38 Binding kinetics of Proteus G spin columns 18 Troubleshooting assistant 44 Protein G affinity resin 21 Glossary 45 Choosing between Protein A and G 23 References 47 Ordering information 48 PROTEIN G 1 proteus 210 G.qxd 5/9/05 1:42 PM Page 2 SECTION 1 Introduction: Affinity purification of monoclonal antibodies has been largely The rapid purification protocols provided in this handbook for confined to the use of Protein A and Protein G affinity chromatography permit the recovery of high levels of chromatography. The Proteus G kit is designed for simple, pure antibodies in minutes. Large numbers of samples can be rapid antibody purification from serum, ascites and tissue processed at the same time. There is negligible hold-up culture supernatant such as those derived from static cultures volume; ensuring high solute recovery with minimal non- and bioreactors. Proteus spin columns replace lengthy and specific absorptive losses. Proteus employs powerful resin- expensive chromatographic methods such as FPLC®. based technology for separating proteins and involves only a few steps, making the isolation of pure antibody samples This Proteus antibody purification kit incorporates pre-packed rapid and simple to perform. Antibody samples purified using Protein G resin plugs in ready-to-use spin columns. The Proteus spin columns may be used in a wide range of objective is to offer the researcher total protein purification laboratory procedures such as 1D or 2D polyacrylamide gel solutions from the initial fractionation stage to the final electrophoresis, Western blotting, ELISA, polishing steps. Resolution of the monoclonal antibody is immunohistochemical or immunofluorescence studies. achieved either in a 2.2 ml centrifuge tube for the Proteus The antibodies are sufficiently pure for radiolabelling, Mini spin column or in a 50 ml centrifuge tube for the conjugations (for example fluorescein) or preparation of Proteus Midi spin column. immunoaffinity columns. FPLC® is a registered trademark of Amersham Biosciences PROTEUS 2 proteus 210 G.qxd 5/9/05 1:42 PM Page 3 SECTION 1 Proteus Benefits: • Proteus G kits are designed to eliminate tedious • The provision of a disposable spin column is ideally suited chromatographic steps normally associated with to GMP production where current user requirements and Protein G chromatography. price sensitivities demand regeneration of the affinity matrix using harsh treatments e.g. peroxyacetic acid • Proteus G reduces time-to-purity by incorporating for Protein A or G resins. protocols to suit various applications, supported by a Proteus Midi Spin Column comprehensive handbook and required buffers in a convenient kit format. Proteus Mini Spin Column • The beaded supports offer excellent flow properties. This combined with the tapering of the spin column provides uniform flow paths that allow optimal use of the available resin bed in swing bucket rotors. • Negligible hold-up volume of the Protein G resin plug ensures high antibody recovery. Proteus Protein G Mini Plug Proteus Protein G Midi Plug PROTEIN G 3 proteus 210 G.qxd 5/9/05 1:42 PM Page 4 MINI Proteus Protein G Mini spin column kit contents: The Proteus Mini kit contains: • 1 x 250 ml 1 x binding buffer (buffer G) bottle. • 16 x Proteus spin column plugs containing Protein • 1 x 125ml 1 x elution buffer (buffer B2) bottle. G-agaroseA-agarose resin. • 1 x 30 ml 1 x neutralization buffer (buffer C) bottle. • 16 x Proteus spin column barrels (0.65 ml capacity in • Plug insertion tool. a fixed angleangle rotor).rotor). • Comprehensive handbook. • 32 x 2.2 mlmicrocentrifuge tubes. • Ultrafiltration spinner instruction sheet. • 16 x 10 kDa MWCO (Polyethersulphone)(Polyethersulphone) • Protocol card. ultrafiltration spinners. NB. PUR015 is a bulk spin column pack containing only Proteus spin column plugs, Proteus spin column barrels, and microcentrifuge tubes and the insertion tool. PROTEUS 4 proteus 210 G.qxd 5/9/05 1:42 PM Page 5 MINI Proteus Protein G Mini spin column kit contents: Neutralization Buffer C Ultrafiltration Spinners Spin Columns Centrifuge Tubes Binding Buffer G Elution Buffer B2 Plug Insertion Tool Resin Plugs PROTEIN G 5 proteus 210 G.qxd 5/9/05 1:42 PM Page 6 SECTION 1 Proteus Protein G Midi spin column kit contents: The Proteus Midi kit contains: • 4 x Proteus spin column plugs containing • 1 x 250 ml 1 x binding buffer (buffer G) bottle. Protein G-agarose resin. • 1 x 125ml 1 x elution buffer (buffer B2) bottle. • 4 x Proteus spin column devices • 1 x 30 ml 1 x neutralization buffer (buffer C) bottle. (20 ml capacity in a swing bucket rotor). • Plug insertion tool. • 8 x 50 ml centrifuge tubes. • Comprehensive handbook. • 4 x 10 kDa MWCO (Regenerated cellulose) • Ultrafiltration spinner instruction sheet. ultrafiltration spinners. • Protocol card. NB. PUR011 is a bulk spin column pack containing only Proteus spin column plugs, Proteus spin column barrels, and centrifuge tubes and the insertion tool. PROTEUS 6 proteus 210 G.qxd 5/9/05 1:42 PM Page 7 SECTION 1 Proteus Protein G Midi spin column kit contents: Ultrafiltration Spinners Spin Columns Neutralization Buffer C Centrifuge Tubes Binding Buffer G Elution Buffer B2 Plug Insertion Tool Resin Plugs PROTEIN G 7 proteus 210 G.qxd 5/9/05 1:42 PM Page 8 SECTION 1 Additional equipment recommended: Storage conditions: • Filters units: 0.2 and 1.2 µm syringe filters for Remove the Proteus Protein G plug box from the kit and clarification. Our recommended filter is the Nalge store it at 2-8˚C. There is no need to place the rest of the kit 50 ml 0.2 µm pore size Supor® Mach V PES conical filter in a fridge or cold room. All buffers, for example, contain (Product Code 564-0020; pack of 12). 0.1 % sodium azide and can be stored at room temperature. • Quartz cuvettes for UV absorbance measurements. Do not freeze the resin plugs or store them at room • UV/VIS spectrophotometer. temperature. Freezing the suspension will damage the • Pasteur pipettes and micro-pipettes. agarose beads. The resin is pre-swollen and de-fined. It is • Marker pen and test tube rack. transported stored in 20% ethanol. Proteus spin columns are • A bench-top centrifuge with swing bucket rotor that can stable for up to 2 years at 2-8 ˚C from the date of accommodate 50 ml centrifuge tubes. manufacture. Expiry date is recorded clearly on the outside of • A microcentrifuge that can accommodate 2.2 ml the pack. microcentrifuge tubes. Disclaimer: N.B. (For the Midi spin column only). The preferred rotor is a swing bucket rotor. For optimal performance with a fixed This product is for research use only and is not intended for angle rotor, ensure that the orientation of the spin column in use in clinical diagnosis. No claims beyond replacement of the rotor is the same for sample binding, washing and elution unacceptable material or refund of purchase price shall be steps. allowed. PROTEUS 8 proteus 210 G.qxd 5/9/05 1:42 PM Page 9 SECTION 1 Technical Support: Contact the Bio-Rad technical support and sales center for assistance: Tel: +44 1865 852 700 NG Fax +44 1865 852 739 E-mail: [email protected] Web: bio-rad-antibodies.com Mail: Endeavour House TEI Langford Lane Kidlington Oxford OX5 1GE UK O PR 9 proteus 210 G.qxd 5/9/05 1:42 PM Page 10 SECTION 1 Specifications: Source: Recombinant Protein G expressed in Typical binding ≥ 20 mg rabbit IgG (Midi) E. coli (N.B. The recombinant Protein G capacity: ≥ 1 mg rabbit IgG (Mini) lacks the albumin-binding domain found ≥ 10 mg rat IgG (Midi) in native Protein G) ≥ 0.6 mg rat IgG (Mini) Supporting Proteus (Note that the Protein G resin can have Covalently coupled to agarose resin matrix: different binding capacities to subtypes of immunoglobulins derived from the Max sample 20 ml (Midi, Swing bucket rotor) same species). volume: 0.65 ml (Mini, Fixed angle rotor) Chemical stability: High Resin bed volume: 1.6 ml (Midi) 0.23 ml (Mini) Endotoxin levels: Unknown Free of Staphyloccocus enterotoxins Ligand density: 2 mg Protein G/ml resin Toxin levels: and hemolysins Bead size range: 45-165 µm Solubility in water: Insoluble Recommended pH 2.0-9.0 Colour coded working pH: Yellow end-caps: PROTEUS 10 proteus 210 G.qxd 5/9/05 1:42 PM Page 11 SECTION 2 Chemical compatibility of the Proteus spin columns Principles of Protein A and G chromatography: All resins are susceptible to oxidative agents. Avoid high This handbook focuses specifically on Protein G affinity temperatures. Protein G is resistant to limited exposure to chromatography. All modes of chromatography can be used 8 M urea pH 10.5 and extremes of pH (eg.