Proteus Protein G Antibody Purification Handbook

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

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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

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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 rtu 1 .x //514 MPage PM 1:42 5/9/05 G.qxd 210 proteus Mail: Web: E-mail: Fax Tel: and salescenterforassistance: Contact theBio-Radtechnicalsupport Technical Support: +44 1865852739 +44 1865852700 UK Oxford OX51GE Kidlington Langford Lane Endeavour House bio-rad-antibodies.com [email protected] 9 SECTION 1 SECTION

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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 ii Antigen to which the monoclonal antibody (iii) Antibody directed against the monoclonal species (ii) A & G. Protein (i) employed: Three types of immunoaffinity ligands are commonly Immuno-affinity separations: 0.1 M NaOH. conditions. However, G is stable to treatment with Protein 1 M NaOH and it is denatured under these aggressive A with respect to pH 11). It lacks the robustness of Protein 8 M urea pH 10.5 and extremes of (eg. 1.0 G is resistant to limited exposure temperatures. Protein All resins are susceptible to oxidative agents. Avoid high Chemical compatibility of the Proteus spin columns was raised. (usually anti-mouse IgG). SECTION 2 remains the principal theme of this handbook. G chromatography therefore, use of anti-antibodies. Protein A and G is widespread has largely superseded the Protein are selected. The use of if the appropriate buffer formulations it lends itself to the bind, wash and elute mode of operation G is extremely attractive as antigens. The simplicity of Protein A or G, immobilized anti-antibodies and Protein techniques include of the protocol is required. Affinity antibodies and different subclasses, a degree of customization exchange chromatography can resolve different polyclonal effectively for the separation of antibodies. Although ion chromatography. All modes of chromatography can be used G affinity This handbook focuses specifically on Protein Principles of Protein A and G chromatography :

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SECTION 2

Application drivers for Protein A and G chromatography: Affinity chromatography:

Monoclonal antibody purification for solution The essence of affinity chromatography utilizes the concept state assays and Western blotting Mini & Midi of bio-specificity, implying an interaction between a natural Semi-preparative antibody purification Midi binding site and the natural ligand, whether it be enzyme- Purification of polyclonal antibodies for solution substrate, enzyme-inhibitor or Protein A/G-antibody state assays and Western blotting Mini & Midi interactions. Removal of endotoxins from an antibody solution Mini & Midi Often a researcher needs to choose the correct base matrix, Small scale antibody purification Mini select the optimal activation chemistry and couple a suitable Screening antibody expression Mini ligand to develop an affinity column to purify the target Immunoprecipitation studies Mini protein. Proteus spin columns remove the guesswork Purification of antibodies for immuno- associated with optimizing the resin chemistry by offering histochemical and immunofluorescence studies Mini ready-to-use resin plugs that have satisfied stringent quality control to guarantee reproducible purification performance. Many affinity support preparations require less than 5 ml resin so these Mini and Midi spin columns contain ideal bed volumes. PROTEUS 12 proteus 210 G.qxd 5/9/05 1:42 PM Page 13

SECTION 2

The only decisions required by the researcher are whether The majority of purifications require fast processing times to they wish to work with Protein A or G resin. Optimal buffer minimise the hydrolytic actions of proteases. Proteus Protein and elution conditions for the purification step of many G kits allow multiple parallel purifications to be achieved common IgG subclasses have been defined and these are without the need to employ expensive PEEK tubing (protein- supplied with the kits. friendly)-based chromatography systems. For example, 12 parallel small scale antibody purifications can be achieved in a Most polymeric mini-columns currently offered operate by microfuge containing a 12 tube fixed angle rotor. gravity flow and typically contain less than 5 ml resin. The columns are normally constructed from polystyrene or Remove top cap A. The top of the polypropylene with a simple barrel and the addition of porous plug is indicated by disks to contain the resin. However, the affinity separation a yellow sticker on the cap as shown. often takes several hours to complete; in addition the TOP researcher usually has to pack the column, which can add a Recessed end minimum 2 further hours to the purification step. The elaborate nature of the packing means that it is usually undertaken by an experienced laboratory worker. B. Unwrap the sealing film from both ends of the Midi plug. The Mini plug has no sealing BOTTOM film. Remove the top and bottom end caps. The top of the plug is recessed. The plug must be inserted into the spin column correctly with recessed end uppermost. Remove bottom cap

Fig. 1. Schematic diagram of a Proteus Mini or Midi spin PROTEIN G column plug. 13 proteus 210 G.qxd 5/9/05 1:42 PM Page 14

14 PROTEUS end uppermost. column with the recessed Place the plug into spin column the plug into spin Loading Top of plug The plug and play concept for the Proteus Mini spin column insertion tool. end of the spin column using plug the plug Push FULLY MINI into the tapered centrifugation. with binding buffer followed by It is now ready for pre-equilibration proteus 210 G.qxd 5/9/05 1:42 PM Page 15 column the plug into spin Loading end uppermost. column with the recessed Place the plug into spin Top of plug The plug and play concept for the Proteus Midi spin column Push the plug Push insertion tool. column using the plug tapered end of the spin FULLY into the MIDI centrifugation. buffer followed by equilibration with binding It is now ready for pre- insertion tool. removed using the plug use, the plug is After spin column plug from the Removing the

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16 PROTEUS Proteus spin column at 4 ˚C. Proteus the target protein in a the need arises, to be able purify importance is the ability to process samples rapidly and, if removed (see delipidation protocol on page 25). Of equal or ascites should also be can be found at high levels in serum Lipids, which sera, ascites and tissue culture supernatants. common during storage and repeated freeze/thaw cycles in Aggregation/precipitation of proteins is chromatographic run. they have been filtered several days before the All samples should be filtered just prior to loading even if particulates that could clog the resin flow channels. sample is first filtered through a G spin column. It is important that the used in the Proteus ascites or an artificial standard can be serum, supernatant, biological extract, a cell culture Any sample, such as a crude conditions for Proteus Protein G spin columns General considerations for selecting optimal binding 0.2 µm filter to remove SECTION 2 1 x binding buffer provided in the kit. separations, the sample should simply be diluted 1:1 (v/v) in exchange A or G chromatographic support. In Protein ultrafiltration before it is applied to an affinity or ion the sample needs to be dialysed or diafiltered by interaction could be weak or non-existent. In many instances, concentration, presence or absence of metal ions etc, the binding conditions are not optimal with respect to pH, salt are critical for successful separation of the protein. If Optimal conditions for binding the target molecule to a resin proteus 210 G.qxd 5/9/05 1:42 PM Page 17 that bind strongly to Protein A or G resin. that bind strongly to Protein capacities can be substantially lower than those antibodies salt concentrations. Note, however, that their binding salt concentration is higher than 1 M, but bind poorly at low A when the and rat IgG2b bind well to immobilized Protein enhancing hydrophobic interactions. Mouse IgG1, rat IgG1 A by reducing severe ionic interactions and Igs to Protein concentration can significantly affect the binding of mouse G is high over a broader pH range. Salt capacity of Protein A is optimal at pH 8-9, whereas the binding for Protein immunoglobulins (Igs) is pH-dependent. The binding capacity G with A or Protein The interaction of immobilized Protein SECTION 2 Choosing the cor separations is: G affinity The typical neutralization buffer used for Protein 0.1 M Sodium acetate pH 3.5 (iii) 0.1-0.2 M Glycine/HCl pH 2.5-3.0 (ii) 0.1 M Sodium citrate pH 3.0 (i) separations are: Typical G affinity elution buffers employed in Protein 20 mM Sodium acetate pH 7.4 (iv) 20-100 mM Sodium phosphate pH 7.0-7.2 (iii) 50 mM Tris, 2.7 mM potassium chloride, (ii 10-100 mM Sodium phosphate, 2.7 potassium (i) separations are: Typical G affinity binding buffers employed in Protein separations: i 1M Tris/HCl pH 9.0 (i) 0.137 M NaCl pH 8.0 (TBS) chloride, 0.137 M NaCl pH 7.4 (PBS) rect buffer conditions for Protein G

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18 PROTEUS * No Proteus Midi Protein A and G performance data is available for centrifugal speeds greater than 1,500 g. A and G performance Midi Protein No Proteus * elution. restored to neutrality after It is therefore critical that the pH antibody structure. and distort the antibody titre, decrease immuno-reactivity harsh elution conditions. Acidic pH is known to reduce the denaturation is caused by target protein. Most observed the integrity and activity of conditions must preserve low pH values. Above all, the elution activity at very monoclonal antibodies) are acid-labile and they can lose their important to appreciate that a few proteins (e.g. some separations involve a reduction in pH to 2.5. It is G affinity The most common elution conditions for Protein Eluting the antibody from a Proteus G spin column: SECTION 2 speed does not exceed 1,500 g*. phase through the column support when centrifugal bed compensates completely for the velocity of mobile Midi resin area of the Proteus surface that the large internal bead volume. Our studies demonstrate to reach the internal move past the beads faster than diffusion time necessary the separation; too fast a flow will cause mobile phase to the column support is inextricably related to efficiency of important in achieving optimal separation. Flow rate through The flow rate through an affinity chromatography support is Binding kinetics of Proteus G spin columns proteus 210 G.qxd 5/9/05 1:42 PM Page 19 effects. diffusion separation can be adversely affected by secondary slow and resolution of the protein chromatography is very Traditionally, bead structure. internal gravity flow ligand to allow for optimal diffusional flow through the kinetics between the protein molecule and immobilized spin column have sufficiently rapid association the Proteus G resin chemistries used in Mini spin columns. The Protein A and G Protein have been achieved with he Proteus mm) 11,960 in a fixed angle rotor with an average radius of 49 Centrifugal speeds as high 13,000 rpm (equivalent to SECTION 2

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20 PROTEUS purifying antibodies are: purifying G affinity purification. The important criteria to consider when The focus of the following section will be devoted to Protein v Antibody Host species and isotype Yield and purity required source (v) Downstream application e.g. immunoassay, immunotags etc Time and cost of the purification (iv) (iii) (ii) (i) Ascites Static Culture Monoclonal 1 mg/ml 0.05 mg/ml Medium serum Ab Medium serum 0.05 mg/ml 1 mg/ml Monoclonal Bioreactor Static Culture Serum Source oolnl1 gm 9 mg/ml 9 mg/ml 10 mg/ml 10 mg/ml Monoclonal Monoclonal oylnl1 gm 1 mg/ml 10 mg/ml Polyclonal Type oa bSeii bContamination Specific Ab Total Ab SECTION 2 Mouse Ab Ab Medium serum Ab Other serum proteus 210 G.qxd 5/9/05 1:42 PM Page 21 Protein G affinity resin: Streptococci G is a cell wall protein from group C and Protein approximately 22 kDa. produced in immunoglobulins. Recombinant G is Protein purifying A or G for researchers use recombinant Protein currently G. Consequently,is removed in recombinant Protein most G has an albumin binding site which addition, native Protein G agarose column. In immunoglobulin from a Protein However, a lower elution pH is required to desorb the A. affinity than Protein G also tends to have a higher antibody binding Protein (PBS). phosphate pH 7.4 with 0.138 M NaCl and 2.7 mM KCl ionic strength. Typically, a researcher will use 10 mM and the binding buffer does not need to have such a high rat IgG2a, IgG2b, human IgG3, bovine IgG1 and sheep in that it binds to a wider range of immunoglobulins such as A G has some benefits over Protein specific for IgG. Protein E. coli that binds strongly to the Fc region of IgG. It is and has a molecular weight of SECTION 2 ✖ ✔ ✔✔ ✔✔✔ ✔✔✔✔ respective antibodies: Key code for relative affinity of Protein A & G for = No affinity No Slight affinity = Weak affinity = Moderate affinity = Strong affinity = =

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22 PROTEUS Rat IgM Rat IgG2c Rat IgG2b Rat IgG2a Rat IgG1 Mouse IgM Mouse IgG3 Mouse IgG2b Mouse IgG2a Mouse IgG1 Human IgM Human IgE Human IgD Human IgA Human IgG4 Human IgG3 Human IgG2 Human IgG1 Binding A ffinities of Protein A and G ✔✔✔✔ ✔ ✔✔✔✖✔✔✖ ✖✔ ✔✔✔✔ ✔✔✔ ✔ ✔✔✔✔ ✔✔ ✖ ✔✔✔ ✖ ✔✔✔✔ ✖ ✔✔✔ ✖ ✔✔ ✔✔ ✔✔✔✔ ✔✔✔✔ ✔✔ ✔✔ ✔✔✔✔ ✔✔✔✔ ✔✔✔✔ ✖ ✔✔✔✔ Protein G ✔✔✔✔ Protein A / / ✖✖ ✖✖ SECTION 2 Human F(ab') Human Fab Fragments Chicken IgG IgG Pig Goat IgG Sheep IgG Bovine IgG IgG Guinea Pig Hamster IgG Rabbit IgG Human Human Human Fc Human scFv λ κ 2 ✔✔ ✔✔✔ ✖✔ ✔✔✔✔ ✔✔✔ ✔ ✔✔ ✔ ✔✔ ✔✔✔ ✔✔✔✔ ✔✔✔ Protein G ✔✔✔✔ Protein A ✔✔✔ ✖✖ ✖✖ ✔✔ ✔✖ ✔✔ / / ✖✔✔✖✔✔ proteus 210 G.qxd 5/9/05 1:42 PM Page 23 need for cleaning. place procedures. However, spin columns obviate the Proteus A withstands harsher conditions used in cleaning-in- Protein G. A followed by Protein experiment first with Protein G, researchers A compared to Protein tend to Protein more difficult. Interestingly, due to the lower cost of of the immunoglobulin making elution and complete recovery However, stronger, G is often the binding of Igs to Protein A. therefore has a wider reactivity profile than Protein G binds to all major Ig classes exceptProtein IgM and subclasses. isolate one particular subclass of IgG from the other to IgG3). For necessary immunological studies, it is often IgG3, and IgG4) in mouse (IgG1, IgG2a, IgG2b subclasses of IgG have been identified in human (IgG1, IgG2, subclasses with different biological properties. Four Immunoglobulin G from most species consists of several Proteus spin columns: Choosing between Protein A and G SECTION 2 complex mixtures is assured. of therapeutic antibodies from such as the selective recovery coupled to careful design of the strategic protocol, objectives binding proteins with the appropriate cross-reactivities for selecting purification tools. By immunoglobulin- processing, the cost and reproducibility are key criteria The needs of the researcher dictate that the speed of sample interaction. manifests itself as a loss of titer and increased non-specific neutralization to reduce the extent of denaturation which elution. Consequently, eluted antibodies require pH 3.0 and human antibodies require at or below 2.5 for concentration is needed. mouse and rat IgG usually requires for method development. No adjustment of pH and salt G binds mouse and rat antibodies without the need Protein

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24 PROTEUS abhdae - - - - 271 -171 -412 9-14 7-11 7-11 7-11 12 2-3 IgE 2-3 IgD immunoglobulins makes it the natural ligand choice for many researchers involved in IgG isolation. 2-3 G for the Fc region of such as those derived from static cultures and bioreactors. The affinity of Protein culture supernatants ascites, or cell Immunoglobulin binding proteins have been used extensively for the purification of IgG molecules from serum, sIgA 2-3 IgA2 Protocol for purif IgA1 % Carbohydrate chain domains IgM Number of heavy of heavy chain IgG4 Molecular weight Molecular weight IgG3 constant IgG2 Sedimentation concentration (mg/ml) IgG1 Mean serum Heavy chain Immunoglobulin Table 1. Physical properties of immunoglobulin subclasses. ying monoclonal antibodies using Proteus Protein G kits 4,0 4,0 7,0 4,0 7,0 6,0 6,0 8,0 8,0 188,000 184,000 385,000 160,000 160,000 970,000 146,000 170,000 146,000 146,000 1005,0 0005,0 5005,0 2005-6006,0 72,500 69,700 52-56,000 52,000 56,000 65,000 51,000 60,000 51,000 51,000 S7 S7 9 S7 1 S8S 7S 11S 7S 7S 19S 7S 7S 7S 7S γ . . . . .500 0.00005 0.03 0.05 0.5 3.0 1.5 0.5 44445 1 444 45 3 9 1 γ 2 γ 3 γ 4 SECTION 3 µ γ 1 α 1 α 1 or α 2 δ 1 ε 1 proteus 210 G.qxd 5/9/05 1:42 PM Page 25 5) Exchange the sample into TBS (Tris Exchange the sample into TBS Buffered Saline) using 5) Discard the precipitate. 4) Centrifuge at 10,000 g for 10 min. 3) Mix for 15 min. 2) Add 0.04 ml 10% dextran sulphate solution and 1 1) lipids and lipoproteins. following protocol is a gentle and easy method for removing antibody solutions which they need to delipidate, the samples derived from ascites fluid. For end users who have presence of lipids and lipoproteins, especially in antibody All protein A & G affinity columns are affected by the Delipidation procedure: as PBS. buffer exchange into a phosphate-containing buffer such dialysis, ultrafiltration or a desalting column. Do not 1 M calcium chloride per ml sample. SECTION 3 Neutralization buffer (Buffer C ): • Elution buffer (Buffer B 2): • Binding buffer (Buffer G ): • All buffers contain 0.1% sodium azide as a preservative. Buffers in the kit: 1 M T ris/HCl buffer pH 9.0 0.2 M Glycine/HCl buffer pH 2.5 0.1 M sodium phosphate buffer, 0.15 NaCl, pH 7.4

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MINI

Step by step protocol for Mini Spin Columns SAMPLE LOADING (Total spin time = 6 mins) RESIN PLUG LOADING 4. Dilute the sample 1:1 (v/v; eg. add 0.5 ml 0.2 µm filtered sample to 0.5 1. Load the pre-packed resin Mini plug containing immobilized ml binding buffer G, pH 7.4). Mix by inverting the capped tube 3-4 recombinant Protein G resin into the barrel of the Proteus spin times. Pipette the 0.65 ml sample into the spin column. Centrifuge the column using the insertion tool as shown on page 14. spin column at 640 g (2,600 rpm in a Heraeus Biofuge Pico or 3,000 rpm in a Sanyo MSE Micro Centaur) for 6 min. PRE-EQUILIBRATION (Total spin time = 2 mins) 2. Equilibrate the Protein G spin column with 0.65 ml binding buffer G, N.B: Increase the spin time or speed if any sample remains above pH 7.4 by centrifuging the spin column at 1,800 g (4,400 rpm in a the plug. Heraeus Biofuge Pico or 5,000 rpm in a Sanyo MSE Micro Centaur) for 1 min*. Repeat this pre-equilibration step with 0.65 ml binding buffer G, pH 7.4 at 1,800 g for 1 min. WASHING (Total spin time = 3 mins) 5. Wash the spin column three times with 0.65 ml binding buffer G, pH 7.4 to remove unbound contaminants by centrifuging the Proteus spin CLARIFICATION OF SAMPLE columns for 1 min at 1,800 g (4,400 rpm in a Heraeus Biofuge Pico or 3. Filter 1 ml sample through a single 0.2 µm syringe filter to remove any 5,000 rpm in a Sanyo MSE Micro Centaur). The unbound wash will cellular debris. contain non-immunoglobulin components.

N.B: Protein precipitation is common during storage and repeated freeze/thaw cycles in ascites, sera and tissue culture supernatants. As with all forms of chromatography, it is important that the sample is filtered through a final 0.2 µm syringe filter immediately before loading it on to the spin column.

* If 1 spin column is to be used, ensure that the spin column is counterbalanced in the microfuge with a microcentrifuge tube filled with the correct level of water. PROTEUS 26 proteus 210 G.qxd 5/9/05 1:42 PM Page 27 ELUTION (Total spin time = 2 mins) .Elute the bound IgG with 0.5 ml elution 6. antibody. you want to recover N.B: Do not pool the two eluate fractions if this elution step. eluate with neutralization buffer C. Repeat tube to ensure thorough mixing of the final spin column for 1 min at 1,800 g. Swirl the approximately 7.5. Centrifuge the Proteus C to bring the pH of sample tube containing 65 µl neutralization buffer buffer B2 directly into a fresh centrifuge concentrated purified Used Spin Column Pure Antibody Pure MINI ANTIBODY PURIFIED THE CONCENTRATING AND DESALTING 7. If necessary, de-salt and concentrate the antibody If necessary, 7. EEEAINO H RTI IIPLUG MIDI G PROTEIN THE OF REGENERATION .Wash the Midi plugs twice with 0.65 ml elution buffer B2 8. in 10-50 % glycerol at -20 ˚C for long term storage. in 10-50 % glycerol at -20 ˚C for long term We recommend freezing the antibodies in small aliquots the antibodies are to be stored at 2-8 ˚C. spinner supplied. Add 0.05-0.2 % w/v sodium azide if ultrafiltration preparation using the 10 kDa MWCO until further use. 0.1 % sodium azide (made up in distilled water) at 2-8 °C stored, without their end caps, in binding buffer G or used immediately. After regeneration, plugs can also be of another bind-wash-elute cycle if the plugs are to be re- to the pre-equilibration step 1,800 g for 1 min. Proceed buffer G (pH 7.4) by centrifuging the spin columns at 1 min. Then wash the plugs twice with 0.65 ml binding (pH 2.5) by centrifuging the spin columns at 1,800 g for

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28 PROTEUS Easy-to-read Mini Purification Protocol E.g. Human ser ah# .5m B H74180g1 min 1 min 1 min 1,800 g 1 min 1 min 1,800 g 1,800 g 1,800 g 1,800 g Spin Time EB2 65 µl NBC BBA pH 7.4 EB2 65 µl NBC RCF BBA pH 7.4 1 min 0.5 ml 6 min BBA pH 7.4 0.65 ml 1 min 0.5 ml 1,800 g 0.65 ml Final Eluate #2 640 g 1,800 g 0.65 ml Final Eluate #1 BBA pH 7.4 1:1 serum: Wash #3 Step Wash #2 BBA pH 7.4 0.65 ml Wash #1 BBA pH 7.4 Volume Sample Loading 0.65 ml #2 Pre-equilibration 0.65 ml #1 Pre-equilibration Fraction um MINI proteus 210 G.qxd 5/9/05 1:42 PM Page 29

MINI

Easy-to-read Mini Regeneration Protocol

Fraction Volume Step RCF Spin Time Clean-up #1 0.65 ml EB2 pH 2.5 1,800 g 1 min Clean-up #2 0.65 ml EB2 pH 2.5 1,800 g 1 min Wash #1 0.65 ml BBA pH 7.4 1,800 g 1 min Wash #2 0.65 ml BBA pH 7.4 1,800 g 1 min PROTEIN G

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30 PROTEUS unit of equal weight (adjusted with distilled water) and without a Protein G resin plug. unit of equal weight (adjusted with distilled water) and without a Protein If 1 spin column is to be used, ensure that the counterbalanced with a * LRFCTO FSAMPLE OF CLARIFICATION PRE-EQUILIBRATION (TotalPRE-EQUILIBRATION spin time = 3 mins) the pre-packed Load resin Midi plug containing immobilized 1. RESIN PLUG LOADING .Filter 12-15 ml sample through a single 1.2 µm (25 mm diameter) 3. 2. Step by step protocol for Midi Spin Columns pH 7.4 by centrifuging the spin column at 500 g for 3 min*. Equilibrate the Protein G spin column with 10 ml binding buffer , loading it on to the spin column. is filtered through a final 0.2 µm syringe filter of chromatography,As with all forms it is important that the sample freeze/thaw cycles in ascites, sera and tissue culture supernatants. precipitation is common during storage and repeated N.B: Protein filter. clarified sample through a single 0.2 µm (25 mm diameter) syringe syringe filter to remove any cellular debris. Then, the partially column using the insertion tool as shown on page 15. spin of the Proteus G resin into the barrel recombinant Protein immediately before MIDI WASHING (Total spin time = 6 mins) LOADING (Total spin time = 30 mins) SAMPLE 4. 5. N. B: Increase the spin time or speed if any sample remains above plug. spin column at 150 g for 30 min. times. Pipette the 20 ml sample into spin column. Centrifuge ml binding buffer G , pH 7.4). Mix by inverting the capped tube 3-4 non-immunoglobulin components. columns for 3 min at 500 g. The unbound wash will contain spin remove unbound contaminants by centrifuging the Proteus Wash the spin column twice with 10 ml binding buffer G , pH 7.4 to Dilute the sample 1:1 (v/v; eg. add 10 ml 0.2 µm filtered to 10 proteus 210 G.qxd 5/9/05 1:42 PM Page 31 ELUTION (Total spin time = 6 mins) .Elute the bound IgG with 10 ml elution 6. purified antibody. if you want to recover N.B: Do not pool the two eluate fractions elution step. neutralization buffer C. Repeat this thorough mixing of the final eluate with min at 500 g. Swirl the tube to ensure spin column for 3 Centrifuge the Proteus pH of the sample to approximately 7.5. neutralization buffer C to bring the centrifuge tube containing 1.3 ml buffer B2 directly into a fresh concentrated Used Spin Column Pure Antibody Pure MIDI ANTIBODY PURIFIED THE CONCENTRATING AND DESALTING 7. If necessary, de-salt and concentrate the antibody If necessary, 7. EEEAINO H RTI IIPLUG MIDI G PROTEIN THE OF REGENERATION .Wash the Midi plugs with 10 ml elution buffer B2 (pH 8. glycerol at -20 ˚C for long term storage. glycerol at -20 ˚C for long term freezing the antibodies in small aliquots 10-50 % antibodies are to be stored at 2-8 ˚C. We recommend spinner supplied. Add 0.05-0.2 % w/v sodium azide if the ultrafiltration preparation using the 10 kDa MWCO further use. sodium azide (made up in distilled water) at 2-8 °C without their end caps, in binding buffer G or immediately. After regeneration, plugs can also be wash-elute cycle if the plugs are to be re-used to the pre-equilibration step of another bind- Proceed 7.4) by centrifuging the spin columns at 500 g for 3 min. Then wash the plugs with 10 ml binding buffer G (pH 2.5) by centrifuging the spin columns at 500 g for 3 min. 0.1 % stored, until

31 PROTEIN G proteus 210 G.qxd 5/9/05 1:42 PM Page 32

32 PROTEUS Easy-to-read Midi Protocol E.g. Human ser Easy-to-read R ah 0m B H7450g3 min Spin Time 3 min 500 g RCF 500 g BBA pH 7.4 3 min EB2 pH 2.5 3 min 10 ml Step 500 g Spin Time 3 min 500 g 10 ml 3 min Volume 500 g 3 min RCF 500 g Wash EB2 1.3 ml NBC BBA pH 7.4 30 min 500 g Clean-up EB2 1.3 ml NBC BBA pH 7.4 Fraction 150 g 10 ml 10 ml BBA pH 7.4 1:1 serum: 10 ml 10 ml Step BBA pH 7.4 Final Eluate #2 Final Eluate #1 20 ml Volume Wash #2 Wash 10 ml #1 Sample Loading Pre-equilibration Fraction egeneration Midi Protocol um MIDI proteus 210 G.qxd 5/9/05 1:42 PM Page 33 can be measured for both affinity and avidity. gels or ELISA. Antigen binding parameters polyacrylamide of reducing or non-reducing SDS- densitometry can be quantified by a standard protein assay, scanning of immunoglobulins kDa bands (Fig. 2, page 33). Recovery IgG appears in a reducing SDS-PAGE as 25 kDa and 50-55 PAGE under reducing or non-reducing conditions. Note that The antibodies can also be monitored for purity by SDS- within a range of 1 mg/ml to 20 sample. ELISA assay to accurately measure antibody concentrations of pure antibody solutions. Most researchers use a sandwich the concentrations and quantitative method for determining absorbance reading. However, they can provide a reliable contaminating proteins can also contribute to the antibody concentrations are only estimates as other multiplying the absorbance at 280 nm by 0.72. These of IgG (mg/ml) in the sample can be measured by law,By using the Beer-Lambert A = ε. c.l, the concentration SECTION 3 with the Proteus Protein G kit purified from (+ foetal calf serum) cell culture supernatant Fig. 2. Reducing gel of mouse IgG1 SDS-Polyacrylamide Albumin Lane 3: Pure mouse IgG1 eluted from the Proteus Protein mouse IgG1 eluted from the Proteus Pure 3: Lane before Hybridoma cell culture supernatant 2: Lane Molecular weight markers. 1: Lane 21.5 kD 116 kD 205 kD 31 kD 45 kD 66 kD 97 kD G spin column spin column. loading on to the Proteus Lane 1 Lane 2 Lane 1 Lane Lane 3 Lane Heavy chain Light chain

33 PROTEIN G proteus 210 G.qxd 5/9/05 1:42 PM Page 34

34 PROTEUS

Recovered Antibody (mg) mg) and the % CV is 0.84 %. (1.22 rabbit serum. All data is within 5 % of mean recovery Mini Fig. 4a. Graph showing that typical IgG capacity of a Protein G 0.2 0.4 0.6 0.8 1.2 1.4 1.6 spin column remains unaffected after 3 re-uses using spin column remains unaffected after 0 1 1.12 123 Number of Re-Uses 1.21 1.32 SECTION 3

Recovered Antibody (mg) mg) and the % CV is 2.4 %. (25.4 rabbit serum. All data is within 5 % of mean recovery Midi Fig. 4b. Graph showing that typical IgG capacity of a Protein G 10 15 20 25 30 35 40 spin column remains unaffected after 5 re-uses using spin column remains unaffected after 0 5 24.8 12345 26.0 Number of Re-Uses 26.4 25.2 24.6 proteus 210 G.qxd 5/9/05 1:42 PM Page 35 bottom of bucket. Value = r. mm from center of rotor to Take radius measurement in running position Swing bucket in MIDI the radius (r) = 160 mm. to 1,670 rpm when Eg. 500 g corresponds RCF = 1.12 x r position). running position (when the bucket is rotated through 90˚ in its of the rotor to bottom swing bucket at its open providing an accurate measurement is taken from the center and RCF (g force). will work on any rotor This formula See the diagram to enable accurate conversion between rpm Many centrifuges display only rpm. reduced performance. those indicated may damage the resin matrix and result in speeds. Use of considerable higher speeds than at the correct spin columns are centrifuged It is important that the Proteus buck How to convert rpm g force (R et rotor ( 1000 rpm ) 2 CF) using a swing

35 PROTEIN G proteus 210 G.qxd 5/9/05 1:42 PM Page 36

MINI

Take radius measurement in mm from center of rotor to How to convert rpm to g force (RCF) using a 45° fixed center of microcentrifuge tube lid. Value = rmin. angle rotor

It is important that the Proteus spin columns are centrifuged max. radius (r) at the correct speeds. Use of considerable higher speeds than rmin those indicated may damage the resin matrix and result in reduced performance. Many centrifuges display only rpm. See the diagram to enable accurate conversion between rpm and RCF (g force). This formula will work on any rotor providing an accurate measurement is taken from the center of the rotor to the centre of the microfuge tube lid.

rpm 2 40 mm RCF = 1.12 x r(1000)

Eg. 640 g corresponds to 3,000 rpm when the max radius (r) = 63.2 mm. (Eg. MSE Micro Centaur microfuge). 1770 g corresponds to 5,000 rpm when the max radius (r) = 63.2 mm. (Eg. MSE Micro Centaur microfuge).

Determination of the maximum radius (r) PROTEUS r = minimum radius in mm (rmin) + 40 mm (the length of the micro-centrifuge tube). sin 45° 36 proteus 210 G.qxd 5/9/05 1:42 PM Page 37 anhydrous M Add 15.0 g glycine (free base; M Elution Buffer B2 (0.2 M Glycine/HCl buffer pH 2.5) distilled water. Add 12.3 g sodium phosphate (dibasic; Na Binding Buffer G (0.1 M sodium phosphate buffer, 0.15 NaCl, pH 7.4) azide as a preservative and can be stored at room temperature: G kit. All buffers contain 0.1 % sodium Protein Use the following recipes to prepare buffers supplied with Proteus volume to 1 L with distilled water. Add 103.72 g T ris base (M Neutralization Buffer C (1 M Tris/HCl buffer pH 9.0) volume to 1 L with distilled water. Protein G kit buffer for r 120.0), 8.77 g NaCl (M mulation: r 121.1), 22.72 g T ris hydrochloride (M r 75.07), 1.0 g NaN r 58.44), 1.0 g NaN 2 HPO 4 ; anhydrous M 3 to 900 ml distilled water. Titrate with HCl to pH 2.5. Make up final 3 APPENDIX to 900 ml distilled water. Make up final volume to 1 L with r 142.0), 1.565 g sodium phosphate (monobasic; NaH r 157.6),1.0 g NaN 3 to 800 ml distilled water. Make up final 2 PO 4;

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APPENDIX

Questions and Answers: 4. What are the typical binding capacities of Proteus Mini and Midi spin columns? 1. What is the preferred rotor for the Proteus Mini and Midi Mini spin columns: Protein A and G resin plugs have typical spin columns? capacities of 1 mg rabbit IgG from serum. Mini spin columns: The preferred rotor is a fixed angle Midi spin columns: Protein A and G resin plugs have rotor. There is no need to orientate the Mini spin column in minimum capacities of 20 mg rabbit IgG from serum. the fixed angle rotor. Midi spin columns: The preferred rotor is a swing bucket 5. How should I prepare my sample for the Proteus spin rotors. For optimal performance with a fixed angle rotor, column? ensure that the orientation of the spin column in the rotor is Many chromatographic procedures demand that the sample is the same for sample binding, washing and elution steps pre-conditioned prior to loading. Protein A affinity separations usually require the sample to be diluted 1:1(v/v) in 1 x 2. Do I need to filter the buffers prepared in my laboratory? binding buffer. We recommend that all samples are diluted It is good laboratory practice to filter all buffers. However, 1:1 (v/v) in the binding buffer supplied with the Protein A or buffers supplied with the kit are pre-filtered for immediate Protein G kit. use.

3. Do I need to pre-filter my sample before loading it on to a Proteus spin column? All samples must be filtered through a 0.2 µm pre-filter PROTEUS immediately before loading the samples on to the spin column. 38 rtu 1 .x //514 MPg 39 Page PM 1:42 5/9/05 G.qxd 210 proteus bucket rotor and up to 10 ml in a fixed angle rotor.fixedangle a in ml 10 to up and bucketrotor columns: spin Midi ml. 0.65 columns: spin Mini column? spin Midi or Mini a to on load can I solution of volume maximum the is What 7. maximum finite a have columns spin Midi and Mini The sample? volume large a process I can How 6. 50% ammonium sulphate is sufficient. sufficient. is sulphate 50% ammonium proteins, other most than sulphate ammonium concentration of lower a at precipitate (γ-globulins) IgGs Although many to concentrateyourtargetantibody.. Otherwise, youcanuseammoniumsulphateprecipitation pump collar accessory (see page 48 for the relevant order code). (> 100-150 ml), we recommend that you use the peristaltic capacity.volume Ifyouhaveavolumeofsample Y ou can load up to 20 ml in a swing a in ml You20 to up load can You can load a maximum volume of Youvolume maximum a load can APPENDIX Midi spin columns: spin Midi steps. spin subsequent in rapidly rehydrate will gel the as columns spin the of performance the affect not will This walls. side the from away shrinkage gel observe may you speed, high very At g. 5,000 than greater speeds spin recommend not do we mm), 49 of radius average an with rotor fixedangle a in rpm (13,000 g 11,960 at tested columns: spin Mini columns? spin Midi and Mini Proteus the spin can I that speed highest the is What 8. low as 50 g. 50 as low as speeds at spun be can devices The columns. spin Midi or Mini the either for speed spin minimum no is There columns? spin Midi and Mini the for speed spin minimum a there Is 9. g. 1,500 than greater speeds centrifugal at available is data performance No g. 1,250 than greater speeds There is no need to spin the devices at devices the spin to need no is There Although the spin columns have been have columns spin the Although

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APPENDIX

10. What are the minimum elution volumes from the Proteus the spin columns at 1,800 g for 1 min. Proceed to the pre- spin columns? equilibration step if plugs are to be re-used immediately. Mini spin columns: The minimum elution volume is 0.5 ml. Do note that spin times of used plugs may be longer. After Midi spin columns: The minimum elution volume is 5 ml. regeneration, plugs can also be stored, without their end caps, in a beaker containing 0.1 % sodium azide (made up in 11. How many times can I re-use the Proteus Mini and Midi distilled water) at 2-8 ˚C until further use. spin columns? Midi spin columns: We recommend that you wash the plugs Mini spin columns: Assuming that all samples are correctly with 10 ml elution buffer B2 (pH 2.5) by centrifuging the filtered, sufficient buffer is provided in the kit for 3 re-uses of spin columns at 500 g for 3 min. Then, re-equilibrate the each Mini spin column. plugs with 10 ml binding buffer G by centrifuging the spin Midi spin columns: Each Midi plug can be re-used typically columns at 500 g for 3 min. Proceed to the pre-equilibration up to 5 times. There is sufficient buffer volume in the kit for step if plugs are to be re-used immediately. Do note that spin one complete use of each Midi spin column. times of used plugs may be longer. After regeneration, plugs can also be stored, without their end caps, in a beaker 12. How can I regenerate the Proteus Protein G plug? containing 0.1 % sodium azide (made up in distilled water) at Mini spin columns: We recommend that you wash the plugs 2-8 ˚C until further use. with 2 x 0.65 ml elution buffer B2 (pH 2.5) by centrifuging the spin columns at 1,800 g for 1 min. Then, re-equilibrate the plugs with 2 x 0.65 ml binding buffer G by centrifuging PROTEUS 40 rtu 1 .x //514 MPg 41 Page PM 1:42 5/9/05 G.qxd 210 proteus plugs. the of performance the upon effect adverse no are There rapidly.rehydrate plugs dried Partially robust. are plugs The steps? centrifugal the during out dry partially plugs the if concerned be I Should 15. The Proteus Protein A or G plugs cannot be autoclaved. be cannot plugs G or A Protein Proteus The plugs? G Protein and A Protein Proteus the autoclave I Can 14. re-use. for stored be then can plugs resin The procedure. regeneration the to continue step, elution the After ligand. A Protein the harm could buffer 2.5 pH in storage Prolonged 2.5. pH buffer elution in columns resin the storing recommend Wenot do step? elution the after immediately column spin the of regeneration perform to need I Do 13. APPENDIX thiocyanate ions. thiocyanate remove to elution after thoroughly dialyzed are they that essential is it ion, chaotropic this using columns, affinity A Protein or columns affinity from eluted are antibodies if and, ions thiocyanate of concentrations low by inhibition to sensitive particularly are tetrachloro-3-6-diphenylglycouril) (1,3,4,6- IODO-GEN or T chloramine either employing activity.reactions of Iodination recovery high and elution rapid ensure will that concentration possible lowest the at agents chaotropic mildest the use to recommended is It columns? affinity from antibodies eluting when use I ion chaotropic of type the with careful be to need I Do 16.

41 PROTEING rtu 1 .x //514 MPg 42 Page PM 1:42 5/9/05 G.qxd 210 proteus 42 PROTEUS high (> 0.1 M) to prevent non-specific binding. non-specific prevent to M) 0.1 (> high quite normally is concentration However,salt exchange. the buffer rapid for tools as serve also can columns spin G or A Protein 5.0. above or to equal be should pH the that except critical, not usually are concentration salt and pH Sample columns? spin Proteus G and A Protein for conditions starting critical the are What 18. storage. before supernatants culture in inhibitors protease of use careful by prevented be often can This action. proteolytic to point may reduction on only visible are that bands protein Other kDa). (25-30 chains light or kDa) (50-55 chains heavy individual the to corresponding seen be will bands more or two 2-mercaptoethanol, or DTT with reduction On kDa. 160-170 about of band protein single a give should antibodies IgG conditions, SDS-PAGEnon-reducing by under analyzed When electrophoresis. gel by measured best is Purity antibodies? isolated the of purity monitor I do How 17. APPENDIX normally be higher than pH 6.0-7.0. pH than higher be normally should pH buffer binding The A. Protein to IgG1 mouse of binding promote may salt high with conjunction in 8-9), (pH pH High subclasses. or antibodies) target from IgG bovine (weakerbinding species different fractionate may 6) pH at (starting steps pH by elution A, ForProtein immunoglobulins. target the desorb to conditions pH acidic more requires usually G Protein variable. critical most the is pH elution The chromatography? G and A Protein during control to parameter important an pH Is 20. IgG1. mouse of binding the promote to pH high with NaCl) M (2-3 salt high use A, Protein with working When adsorption. non-specific reduce to salt M 0.1-0.5 Use chromatography? G and A Protein during concentrations salt the control to need I Do 19. rtu 1 .x //514 MPg 43 Page PM 1:42 5/9/05 G.qxd 210 proteus about loss of activity of acid-labile immunoglobulins. acid-labile of activity of loss about concern is there if pH acidic replace sometimes can M 1 Concentrations of divalent cations (particularly Mg (particularly cations divalent of Concentrations cations? divalent using column spin G and A Protein a from antibodies elute I Can 21. 2+ ) up to up ) APPENDIX

43 PROTEING proteus 210 G.qxd 5/9/05 1:42 PM Page 44

44 PROTEUS The centrifugal speed for the sample loading step can be • If the spin columns are not stored at 2-8 ˚C, or they • the The resin is clogged with particulates. Pre-filter • The sample does not flow easily through the spin column The spin column has been stored at a cool temperature • Bubbles or cracks appear in the resin bed T roubleshooting assistant: increased to a maximum 1,500 g. column may restrict flow through the resin plug. absence of a bacteriostat, microbial growth in the have been used more than once and stored in the procedure. refer to page 25 for the recommended delipidation column. Ascites must be delipidated before use. Please spin sample just before loading it on to the Proteus slowly to room temperature before use. be warmed spin columns should up. Proteus and then rapidly warmed APPENDIX The sample may also need to be filtered carefully. • The sample volume or concentration may be too large • Poor resolution of the target protein • The binding of antibodies to Protein A or G is attributed The binding of antibodies to Protein • of target protein is low The recovery The elution conditions are too mild to desorb • It is The pH of the elution buffer may be incorrect. • column No elution of the target from protein the spin is observed the sample load or volume. for the capacity of resin plug. In this case, reduce reduce the strength of all hydrophobic in part to hydrophobic forces. Use chaotropic salts to the target protein. advisable to prepare new solutions. interactions. proteus 210 G.qxd 5/9/05 1:42 PM Page 45 • The buffer pH and ionic strength is incorrect and The buffer pH and ionic strength is incorrect • or lipids may have precipitated in the resin bed. Proteins • The sample load may be different from the original • The nature of the sample may have altered and so it • The elution profile cannot be reproduced The column may be dirty. spin All claims made of Proteus • There may be hydrophobic interactions between the • There may be an ionic interaction between the protein • The target protein elutes at an unexpected position new buffers will need to be prepared. Use elution conditions which stabilize the sample. parameters constant. sample load. It is advisable to keep all these may be important to prepare a fresh sample. only. columns are guaranteed for the first bind-wash-elute cycle organic solvents. salt concentration and add suitable detergents or resin. In this instance, reduce the sample and Proteus above 50 mM. resin. Youand Proteus should maintain the ionic strength APPENDIX Glossar column volume or CV. chromatographic packed to as the bed. It is also referred bed volume monoclonal antibodies from mice. cell line into the peritoneal cavity. It is a common source of ascites antibodies to a particular antigen. immunized or exposed to an immunogen and contains antiserum antibody. antigen substance. system of vertebrates in response to exposure a foreign antibody binding site on a macromolecule. on a specific interaction between an immobilized ligand and affinity chromatography - a liquid tumour formed by injection of a hybridoma - a liquid tumour formed - a molecule which can bind specifically to an y - an immunoglobulin produced by the immune : - the serum fraction from an animal that has been - the serum - the total volume occupied by - chromatographic separation based

45 PROTEIN G proteus 210 G.qxd 5/9/05 1:42 PM Page 46

46 PROTEUS amino acid composition and carbohydrate content. lgD and IgE. They differ from each other in size, charge, IgA, higher animals. Classes called IgG (the most common), IgM, immunoglobulin (Ig) linkages. immobilized indefinitely in cell culture. reproducing cells that produce specific monoclonal antibodies producing cells with myeloma to generate immortal hybridoma water molecules. Examples include urea and guanidine. of the ordered structure phobic interactions by disrupting chaotropic agent tissue media components and the secreted target. containing (either roller bottle, suspension or perfusion) cell culture supernatant - a hybrid cell line produced by fusing antibody - bound to a surface, usually through covalent - bound to a surface, - a molecule which interferes with hydro- - a molecule which interferes - comprising 5 distinct classes in most - the fluid made during cell culture APPENDIX ion exchange chromatography immunoglobulins. bacteria which specifically bind to the Fc region of protein A/protein G are almost always polyclonal. antiserum immunogen by different cell types. Antibodies from polyclonal antibodies cell line. from a hybridoma of immune cells. They are usually formed monoclonal antibody charge. isoelectric point based on different charge properties of macromolecules. - the pH at which protein has no net - cell wall proteins of certain pathogenic - an antibody derived from a single clone - antibodies produced to the same - chromatographic separation proteus 210 G.qxd 5/9/05 1:42 PM Page 47 ISBN: 0-443-02912-1. 1985 (I Roitt, Ltd., J Brostoff, D Male) Gower Medical Publ. Immunology ISBN: 0-7167-2009-4. WH Freeman and Co. 1995 (L Stryer) Biochemistry, 4th Edition ISBN: 0-9653515-9-9. 1996 (P Gagnon) Validated Biosystems, USA, Purification Tools for Monoclonal Antibodies Co.Publ. ISBN: 0-8053-3931-0. 1996 (Mathews, CK & van Holde, KE) Benjamin/Cummings Biochemistry, 2nd Edition ISBN: 0-19-963550-1. University Press. 1997 (Matejtschuk, P Ed.) IRL PRESS at Oxford Separations: A Practical Approach Affinity R eferences: APPENDIX 1997 (RS Tuan ISBN: 0-89603-400-3. Ed.) Humana Press, Recombinant Protein Protocols 1994 (J Walker ISBN: 0-89603-269-8. Ed.) Humana Press, Basic Proteins and Peptide Protocols ISBN: 0-19-963023-2. Oxford University Press. 1995 (E.L.V. and S. Angal Eds.) IRL PRESS at Harris Protein Purification Applications: A Practical Approach

47 PROTEIN G proteus 210 G.qxd 5/9/05 1:42 PM Page 48

APPENDIX

Ordering Information:

Spin Column Kits Quantity Product Code Accessories Quantity Product Code Mini Protein A Sample Kit 1 PUR006 Protein A Buffer Pack 1 PUR004 (2 A Spin Columns) Protein G Buffer Pack 1 PUR009 Mini Protein G Sample Kit 1 PUR014 Peristatic Pump Collar 1 PUR027 (2 G Spin Columns) Mini Protein A & G Starter Pack 1 PUR028 (2 A & 2 G Spin Columns) Mini Protein A Kit (16 units) 1 PUR008 Mini Protein G Kit (16 units) 1 PUR016 Mini Protein A Bulk Pack (48 units) 1 PUR007 Mini Protein G Bulk Pack (48 units) 1 PUR015 Midi Protein A Kit (4 units) 1 PUR003 Midi Protein G Kit (4 units) 1 PUR012 Midi Protein A Bulk Pack (12 units) 1 PUR002 Midi Protein G Bulk Pack (12 units) 1 PUR011 PROTEUS 48 IMAC cover 4/2/04 9:11 AM Page 2

Bio-Rad Laboratories, Endeavour House, Langford Lane, Kidlington, Oxfordshire, OX5 1GE, UK

Tel: +44 1865 852700 Fax: +44 1865 373 899

Email: [email protected]

Web site: www.bio-rad-antibodies.com

IFUPUR Proteus Protein G Handbook. Issue 5. 05.2016