DOCSLIB.ORG

Identification and Grafting of a Unique Peptide-Binding Site in The

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Identification and Grafting of a Unique Peptide-Binding Site in The Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies Joshua M. Donaldsona,b,1, Cindy Zera,1, Kendra N. Averya, Krzysztof P. Bzymeka, David A. Hornea, and John C. Williamsa,2 aDepartment of Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, CA 91010; and bDepartment of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, PA 19107 Edited* by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved September 19, 2013 (received for review April 18, 2013) Capitalizing on their extraordinary specificity, monoclonal anti- enhance imaging reduces the valency and, thus, affects both the bodies (mAbs) have become one of the most reengineered classes affinity and the tissue specificity compared with the original of biological molecules. A major goal in many of these engineering mAb. In other cases, as part of a pretargeted imaging protocol to efforts is to add new functionality to the parental mAb, including enhance detection, mAbs conjugated with streptavidin or multi- the addition of cytotoxins and imaging agents for medical applica- ple Fabs/scFvs stitched together through a scaffold (e.g., “lock- tions. Herein, we present a unique peptide-binding site within the and-dock”; ref. 12) can be immunogenic (13), unstable in serum central cavity of the fragment antigen binding framework region of (14), and technically difficult and expensive to produce, especially the chimeric, anti-epidermal growth factor receptor mAb cetuximab. at scales necessary for clinical use (15, 16). Although significant We demonstrate through diffraction methods, biophysical studies, advances in antibody engineering have been achieved, practical and sequence analysis that this peptide, a meditope, has moderate challenges remain, many requiring alternative approaches. affinity for the Fab, is specific to cetuximab (i.e., does not bind to Herein, we present a unique interaction that has the potential fi human IgGs), and has no significant effect on antigen binding. We to overcome some of the dif culties in mAb engineering. We fi further demonstrate by diffraction studies and biophysical methods identi ed a binding site for a small peptide in the center of the that the meditope binding site can be grafted onto the anti-human Fab cavity of cetuximab via diffraction studies. Because of the position of the binding interface, we have named the peptide epidermal growth factor receptor 2 mAb trastuzumab, and that the “ ” medius topos antigen binding affinity of the grafted trastuzumab is indistinguish- meditope ( , middle; , place). We demonstrated that the meditope does not interfere with antigen binding. Based on able from the parental mAb. Finally, we demonstrate a bivalent fi meditope variant binds specifically and stably to antigen-bearing structural and biophysical analysis, we identi ed key residues cells only in the presence of the meditope-enabled mAbs. Collec- within the meditope binding site of the Fab and observed that the fi combination of these residues is absent in other clinically relevant tively, this nding and the subsequent characterization and engi- fi neering efforts indicate that this unique interface could serve as mAbs, including a humanized mAb framework. To con rm the structural model and determine whether we could enable medi- a noncovalent “linker” for any meditope-enabled mAb with applica- tope binding in other mAbs, we grafted selected residues onto tions in multiple mAb-based technologies including diagnostics, im- trastuzumab, a humanized anti-HER2 mAb, and demonstrated aging, and therapeutic delivery. by diffraction and biophysical studies that the meditope binds to this meditope-enabled trastuzumab in a similar manner and af- protein engineering | molecular recognition | cancer finity compared with cetuximab. Finally, we created a bivalent analog of the meditope to demonstrate that this interaction can onoclonal antibodies (mAbs) are indispensable tools in be used to specifically target antigen-bearing cells in the presence Mresearch laboratories and have become a central compo- of a meditope-enabled mAb that binds to the specific antigen. nent in modern medicine. Thousands of antibodies are routinely Overall, these results establish the basis for an alternative mAb used in research to detect and/or label specific proteins in a va- platform that offers unique opportunities to improve mAb-based riety of settings. In recent years, dozens of mAbs that block delivery systems for cancer and other diseases. signaling pathways, sequester growth factors, and/or induce an immune response have been successfully implemented in the Significance clinic to treat cancer and other diseases, with hundreds still in active development (1, 2). Antibodies are being reengineered to fi fi The identi cation and subsequent grafting of a unique peptide best capitalize on their extraordinary ligand speci city to add binding site within the Fab domain offers a unique means of new functionalities for a broad range of applications, mostly for adding functionality to monoclonal antibodies through a non- clinical use, such as antibody-drug conjugates (ADCs), antibody- covalent interaction including improved pretargeted imaging, directed enzyme prodrug therapies (ADEPT), immune system alternative payload delivery, and cross-linking of mAbs on cell fi engagement [e.g., Fc modi cations (3), chemokine fusion, bispe- surfaces to enhance their therapeutic potential. cific T-cell engagers (4)] and disease imaging (e.g., immunoPET and pretargeted radionuclide imaging; refs. 5 and 6). Author contributions: J.M.D., C.Z., K.N.A., and J.C.W. designed research; J.M.D., C.Z., K.N.A., Invariably, these engineering efforts have been achieved either K.P.B., and J.C.W. performed research; J.M.D., C.Z., D.A.H., and J.C.W. contributed new through posttranslational chemical modifications or manipula- reagents/analytic tools; J.M.D., C.Z., K.N.A., K.P.B., and J.C.W. analyzed data; and J.M.D., tion of the gene sequence (7–9). Many of the chemical mod- C.Z., K.N.A., K.P.B., and J.C.W. wrote the paper. ifications lead to undesirable consequences that are detrimental Conflict of interest statement: D.A.H. and J.C.W. have founded Meditope Biosciences, to therapeutic and imaging applications (10). For example, to a company based on some of the observations presented here. create ADCs to target cytotoxins to disease sites with the mAb, *This Direct Submission article had a prearranged editor. the chemical conjugation of the toxin (typically involves lysines, Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, reduced cysteines, or sugars on the mAb) produces a heteroge- www.pdb.org (PDB ID code 4GW1, 4GW5, 4HKZ, 4HJG, and 4IOI). neous mixture, which can adversely affect the specificity and 1J.M.D. and C.Z. contributed equally to this work. stability of the mAb and alter its biodistribution (11). Reducing 2To whom correspondence should be addressed. E-mail: [email protected]. the size of the mAb to Fab fragments [e.g., single-chain Fab This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. variable domain (scFvs)] to facilitate tumor penetration and 1073/pnas.1307309110/-/DCSupplemental. 17456–17461 | PNAS | October 22, 2013 | vol. 110 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1307309110 Downloaded by guest on September 27, 2021 meditope-Fab interface, ∼850 Å, is similar to the that of protein A, G, and L. (23) (SI Appendix, Table S2). As an additional confirmation of the structural model, we identified key residues on the meditopes that enabled the in- teraction with the Fab based on the superposition of the cQFD-Fab and cQYN-Fab structures. We observed that the hydrophobic side chains of Phe/Tyr3, Leu5, and Leu10 of both meditopes were positioned nearly identically, suggesting that they make important contacts to the Fab (SI Appendix,Fig.S1). The importance of these residues is confirmed by SPR experiments showing alanine mutations at Phe3, Leu5, or Leu10 of the cQFD meditope resulted in a 5- to 140-fold decrease in affinity (SI Ap- pendix,TableS3). We also observed that the guanidinium group of Arg8 of cQFD forms a hydrogen bond to the backbone carbonyl of Gln111 of the Fab heavy chain. Accordingly, Arg8Ala substitution decreased the affinity by 325-fold (SI Appendix, Table S3). The extended conformation of Arg8 is sterically blocked by the hy- droxyl group of Tyr3 of the cQYN meditope, which causes a backbone rotation and eliminates the Arg8–Gln111 interaction (SI Appendix, Fig. S1). The loss of this interaction partially Fig. 1. Meditope binds to cetuximab Fab framework. (A) Ribbon repre- accounts for the nearly 10-fold difference in the binding affinity sentation of cetuximab Fab (light chain, blue-white; heavy chain, white) with between the two meditopes (Fig. 2A and SI Appendix,TableS3) a stick representation of the cyclic CQFDLSTRRLKC meditope (green). (B) Thus, although the point mutations agree with the structural Electron density maps of cQFD meditope, in stereo and contoured at 1σ.(C) Superposition of the cetuximab-EGFR (1YY9) structure to Fab framework ligands: protein L (1HEZ), protein A (1DEE), protein G (1QKZ), and cQFD meditope (4GW1). Results Identification of the Meditope Binding Site on Cetuximab Fab Framework. In our efforts to create tumor activated antibodies (17), we cocrystallized two peptides, CQFDLSTRRLKC (cQFD) and CQYNLSSRALKC (cQYN), individually with the Fab do- main of cetuximab. These peptides were originally identified by phage display (18) and thought to mimic the tumor epitope EGFR domain III. The initial diffraction data indicated that the unit cell dimensions of each cocrystal were the same as that of the apo cetuximab Fab [Protein Data Bank (PDB) ID code 1YY8; ref. 19]. Because the complementarity determining region (CDR) loops in the published structure make extensive lattice contacts, the data suggested that the peptides were either absent or bound to a different region of the Fab.
Load more

Recommended publications
  • Pierce™ Chromatography Cartridges Protein L
    INSTRUCTIONS Pierce ™ Chromatography Cartridges Protein L 1971.1 89928 89929 Number Description 89928 Pierce Chromatography Cartridges Protein L, 2 × 1mL 89929 Pierce Chromatography Cartridges Protein L, 1 × 5mL Binding Capacity: 4-5mg human IgG/mL of resin bed Note: Protein L is immobilized on crosslinked 6% beaded agarose supplied in 0.05% sodium azide in water. Each product is supplied with an accessory pack (1 female Luer-Lok™ Adapter, 1 connector fitting, 1 column plug and 1 or 2 bottom caps). Storage: Upon receipt store at 4-8°C. Product is shipped at ambient temperature. Do not freeze. Introduction The Thermo Scientific™ Pierce™ Chromatography Cartridges Protein L are convenient, ready-to-use prepacked devices for isolation and purification of immunoglobulin classes IgG, IgM, IgA, IgE and IgD via their kappa light chains. Protein L binds to certain subtypes of antibody kappa light chains, predominant in humans and mice, without interfering with antigen binding sites. Typically, Protein L cartridges are used for purifying monoclonal antibodies from ascites or cell culture supernatants. Protein L does not bind bovine antibodies, eliminating contamination from bovine immunoglobins when purifying serum- supplemented cell culture supernatants. Protein L also binds single chain variable fragments (scFv) and Fab fragments, unlike Protein A, Protein G or Protein A/G. Pierce Chromatography Cartridges are compatible with the major automated liquid-chromatography systems or for manual syringe processing (see Table 1 for general properties of the cartridges). The cartridges attach directly to ÄKTA™ or FPLC Systems without additional connectors. An accessory pack, included with each product, readily adapts columns for use with Luer-Lok Syringe Fittings or 1/16” tubing.
    [Show full text]
  • Pierce Protein L Agarose
    INSTRUCTIONS ® Pierce Protein L Agarose 20510 20512 0778.5 Number Description 20510 Pierce Protein L Agarose, 2 ml settled resin 20512 Pierce Protein L Agarose, 10 ml settled resin Support: Crosslinked 6% beaded agarose supplied as 50% slurry (e.g., 2 ml of settled resin is equivalent to 4 ml of 50% slurry) containing 0.02% sodium azide Binding Capacity: ~5-10 mg human IgG/ml of resin Storage: Upon receipt store product at 4-8°C. Product is shipped at ambient temperature. Table of Contents Introduction .................................................................................................................................................................................1 Important Product Information ....................................................................................................................................................1 Gravity-flow Column Procedure for Antibody Purification ........................................................................................................2 A. Additional Materials Required ......................................................................................................................................2 B. Immunoglobulin Purification Procedure .......................................................................................................................2 Troubleshooting...........................................................................................................................................................................3 Additional Information
    [Show full text]
  • Pierce Protein L Agarose
    INSTRUCTIONS ™ Pierce Protein L Agarose 0778.6 20510 20512 Number Description 20510 Pierce Protein L Agarose, 2mL settled resin 20512 Pierce Protein L Agarose, 10mL settled resin Support: Crosslinked 6% beaded agarose supplied as 50% slurry (e.g., 2mL of settled resin is equivalent to 4mL of 50% slurry) containing 0.02% sodium azide Binding Capacity: ~5-10mg human IgG/mL of resin Storage: Upon receipt store product at 4-8°C. Product is shipped at ambient temperature. Table of Contents Introduction ................................................................................................................................................................................. 1 Important Product Information .................................................................................................................................................... 1 Gravity-flow Column Procedure for Antibody Purification ........................................................................................................ 2 A. Additional Materials Required ...................................................................................................................................... 2 B. Immunoglobulin Purification Procedure ....................................................................................................................... 2 Troubleshooting ........................................................................................................................................................................... 3 Additional
    [Show full text]
  • Protein L—More Than Just an Affinity Ligand
    processes Review Protein L—More Than Just an Affinity Ligand Stefan Kittler 1,2, Mihail Besleaga 1, Julian Ebner 1,2 and Oliver Spadiut 1,* 1 Research Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorfer Strasse 1a, 1060 Vienna, Austria; [email protected] (S.K.); [email protected] (M.B.); [email protected] (J.E.) 2 Alfred Gruber GmbH, Nordstrasse 6, 5301 Eugendorf, Austria * Correspondence: [email protected]; Tel.: +43-1-58801-166473 Abstract: In the past 30 years, highly specific drugs, known as antibodies, have conquered the biopharmaceutical market. In addition to monoclonal antibodies (mAbs), antibody fragments are successfully applied. However, recombinant production faces challenges. Process analytical tools for monitoring and controlling production processes are scarce and time-intensive. In the downstream process (DSP), affinity ligands are established as the primary and most important step, while the application of other methods is challenging. The use of these affinity ligands as monitoring tools would enable a platform technology to monitor process steps in the USP and DSP. In this review, we highlight the current applications of affinity ligands (proteins A, G, and L) and discuss further applications as process analytical tools. Keywords: monoclonal antibodies; antibody fragments; affinity ligands; process analytical technol- ogy; protein A; protein G; protein L Citation: Kittler, S.; Besleaga, M.; Ebner, J.; Spadiut, O. Protein L—More 1. Introduction Than Just an Affinity Ligand. Processes 2021, 9, 874. https:// In 1986, a new type of drug was approved and introduced to the biopharmaceutical doi.org/10.3390/pr9050874 market—the first monoclonal antibody (mAb) with the trade name Orthoclone (OKT3), preventing rejection after kidney transplantation [1].
    [Show full text]
  • Protein L Biosensors | Fortebio
    Protein L Biosensors For whole molecule antibody and antibody fragment quantitation Key features binding to the Protein L Biosensor alters the properties of light on the biosensor surface, allowing the association event to be • Broader recognition of Ig isotypes and species than Protein monitored in real time using the Octet system. Higher antibody A or G concentrations cause faster binding rates. Analyte concentra- • Regenerable and cost-effective format tion is extrapolated from a standard curve. Due to the high specificity of Protein L for the antibody light chain, Protein L bio- • Binds whole molecule Ab and FAb fragments sensors can directly quantitate analytes in conditioned media • Quantitate in crude matrices and complex matrices. Overview Antibody Protein L Protein A Protein G Dip and Read™ Protein L (ProL) biosensors provide a rapid and Rat Total IgG s w m direct method for quantifying a broad set of kappa light chain Rat IgG1 s w m containing immunoglobulins, including whole molecules, FAb Rat IgG2a s nb s fragments and single chain variable fragments, in buffer, condi- tioned media or complex matrices. Protein L, which is factory- Rat IgG2b s nb w immobilized onto the biosensor, binds antibodies through the Rat IgG2c s s s kappa light chain and recognizes a wider range of antibody Rabbit IgG w s s classes than either Protein A or Protein G, including IgG, IgM, Mouse IgG1 s w m IgA, IgE and IgD. Due to its specificity, Protein L recognizes Mouse IgG2a s s s mouse VkI (but not VkII, VkIII and VkIV) and human VkI, VkIII Mouse IgG2b s s s and VkIV (but not VkII).
    [Show full text]
  • 1 Biophysical Enhancement of Protein Therapeutics and Diagnostics
    Biophysical Enhancement of Protein Therapeutics and Diagnostics Through Engineered Linkers Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nicholas Emerson Long Graduate in The Ohio State University Biochemistry Program The Ohio State University 2018 Dissertation Committee Dr. Thomas Magliery, Advisor Dr. Christopher Jaroniec Dr. Edward Martin Jr. Dr. Richard Swenson 1 Copyrighted by Nicholas Emerson Long 2018 2 Abstract Proteins play a major role in virtually every biological process. Thus, proteins are an ideal platform for the next generation of therapeutics. Over the last few decades, technological and scientific advances in protein production and engineering have led to a new wave of protein-based biologics used in clinical settings. In this body of work, we have engineered both a protein-based cancer diagnostic and an immunotherapeutic. Antibody-based biologics are becoming one of the most widely approved drug platforms and owe their success to their versatility in binding targets, high stability, and low toxicity. The anti-TAG-72 cancer-targeting antibody, 3E8, is one such molecule that shows great potential as a diagnostic. We have designed and biophysically characterized a library of 3E8 single chain antibody fragments (scFV) with varying linker composition and length as well as domain orientations. In this library, we have found substantial variation in protein stability, binding affinity, and oligomeric states. Surprisingly, a drastic difference in the oligomeric state of these constructs was seen between conventional IMAC purification and Protein L purification. Therefore, the literature rules for scFV linker design must be updated to include the dependencies on purification method.
    [Show full text]
  • Columns and Resins for Antibody Purification and Immunoprecipitation
    Columns and resins for antibody purification and immunoprecipitation Selection guide Introduction to antibody purification and immunoprecipitation What is antibody purification? Affinity ligands for antibody purification Polyclonal antibodies, monoclonal antibodies (mAb), and Protein A and protein G antibody fragments are usually purified by affinity While protein A and protein G affinity resins are similar in many chromatography. Resins containing an immobilized ligand respects, their specificities for immunoglobulin G (IgG) differ. (e.g., protein A, protein G, or protein L) are used to capture antibodies and antibody fragments. Protein G affinity resin is a good first choice for general purpose capture of antibodies at laboratory scale since it binds a Affinity purification offers high selectivity. Purity levels broader range of IgG from eukaryotic species and also binds above 95% are often possible in just one step. to more subclasses of IgG than Protein A (see Table 1). The protein A ligand is routinely used in pharmaceutical processes and is commonly preferred when purifying human monoclonal antibodies. Protein L Protein L binds to the variable region of the kappa light chain. Therefore, protein L affinity resin is suitable for capture of a wide range of antibody fragments. Fv Heavy chain Light chain Protein L ligand Fab Variable region Constant region Protein A ligand Protein G ligand How does antibody purification work? Fc Kappa and Antibody affinity chromatography is based on the high lambda ligands affinity and specificity to affinity ligands. The binding of an antibody to the ligand is reversible, and IgG the antibody is often eluted by lowering the pH. Fig 2.
    [Show full text]
  • Inactivates Neutrophil Proteins S100A8/A9 Bacterial Surface
    Bacterial Surface Protein L Binds and Inactivates Neutrophil Proteins S100A8/A9 Bo Åkerström and Lars Björck This information is current as J Immunol 2009; 183:4583-4592; Prepublished online 14 of September 28, 2021. September 2009; doi: 10.4049/jimmunol.0901487 http://www.jimmunol.org/content/183/7/4583 Downloaded from References This article cites 57 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/183/7/4583.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Bacterial Surface Protein L Binds and Inactivates Neutrophil Proteins S100A8/A91 Bo Åkerstro¨m2 and Lars Bjo¨rck2 Finegoldia magna is an anaerobic bacterial species that is part of the normal human flora on all nonsterile body surfaces, but it is also a significant opportunistic pathogen causing a wide range of infections.
    [Show full text]
  • Antibody Fragments and Their Purification by Protein L Affinity Chromatography
    Antibodies 2015, 4, 259-277; doi:10.3390/antib4030259 OPEN ACCESS antibodies ISSN 2073-4468 www.mdpi.com/journal/antibodies Review Antibody Fragments and Their Purification by Protein L Affinity Chromatography Gustav Rodrigo 1,†,*, Mats Gruvegård 1,† and James M. Van Alstine 2,3,† 1 GE Healthcare Life Sciences, Uppsala 75184, Sweden 2 JMVA Biotech, Stockholm 11254, Sweden 3 Industrial Biotechnology, Royal Institute of Technology (KTH), Stockholm 10691, Sweden † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: [email protected]. Academic Editor: Satoshi Ohtake Received: 29 June 2015 / Accepted: 27 August 2015 / Published: 11 September 2015 Abstract: Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteria—suggesting its consideration as a key unit operation in antibody fragment processing. Keywords: affinity; antibody fragment; bioprocess; chromatography; monoclonal antibody; protein A; protein L Antibodies 2015, 4 260 1.
    [Show full text]
  • Application Note: Captureselect Affinity Ligands for Antibody Detection and Characterization
    APPLICATION NOTE 9 CaptureSelect affinity ligands for antibody detection and characterization Pim Hermans, Gerwin Grit and Sven Blokland, BAC BV Introduction Antibody purification BAC’s CaptureSelect affinity ligands provide a unique group Using Protein A, Protein G, and Protein L for antibody purifi- of molecules that can be designed for almost any purification cation can be highly effective. However, there are gaps in the target, with as narrow or as broad specificity as is required. The functionality that they provide, and when a target protein falls fragments are derived from the unique heavy chain antibod- into one of these gaps the process of finding an alternative puri- ies found in Camelidae. Heavy chain antibodies are devoid of fication strategy can be extremely frustrating. The main binding the entire light chain and CH1 domain found in conventional characteristics of Proteins A, G, and L are listed below: antibodies (Figure 1). Without the light chain, antigens are bound only by the variable domain of the heavy chain (VHH), without a PROTEIN A AND G loss in binding affinities compared to conventional antibodies. • Bind to IgG Fc domain (CH2-CH3 interface) The VHH domain is the smallest functional antigen-binding • Bind IgG of many species including bovine and mouse (not fragment known that shows high affinity and stability. The equiv- human specific) alent fragment from conventional antibodies is the Fv domain, • Protein A binds human IgG1, 2 and 4 but not IgG3; Protein G which consists of the heavy chain variable domain and the light binds all sub-types chain variable domain held together by a non-covalent bond.
    [Show full text]
  • Protein L-Agarose Immunoprecipitation Reagent: Sc-2336
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . Protein L-Agarose Immunoprecipitation Reagent: sc-2336 PRODUCT • Pellet beads by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4° C. Transfer supernatant (cell lysate) to a fresh 15 ml Protein L is provided as an agarose conjugate for use in immunoprecipitation conical centrifuge tube on ice. only. The product is provided as 0.5 ml agarose in 2.0 ml PBS buffer with 0.02% azide. Protein L-Agarose is pre-blocked with BSA to reduce non-spe - • Transfer 1 ml of the above cell lysate, or approximately 100–500 µg cific immunoglobulin binding. Sufficient product is provided for 100 immuno- total cellular protein, to a 1.5 ml microcentrifuge tube. Add 1–10 µl precipitation reactions, to be used at 20 µl resuspended volume per reaction. (i.e., 0.2–2 µg) primary antibody (optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4° C. REFERENCE • Add 20 µl of resuspended volume of Protein L-Agarose. Cap tubes and 1. Kastern, W., et al. 1992. Structure of peptostrepto-coccal protein l and incubate at 4° C on a rocker platform or rotating device for 1 hour to identification of a repeated immunoglobulin light chain- binding domain. J. overnight. Biol. Chem. 267: 12820-12825. • Collect immunoprecipitates by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4° C. Carefully aspirate and discard radioactive SPECIFICITY supernatant. Protein L-Agarose is suitable for immunoprecipitation of mouse, rat and • Wash pellet 4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less human IgG, mouse, rat and human IgM, IgE and IgA proteins and scFv and stringent), each time repeating centrifugation step above.
    [Show full text]
  • Pierce Protein L Plus Agarose
    INSTRUCTIONS ™ Pierce Protein L Plus Agarose 0777.5 20520 Number Description 20520 Pierce Protein L Plus Agarose, 2mL of settled resin Support: Crosslinked 6% beaded agarose supplied as a 50% slurry (e.g., 2mL of settled resin is equivalent to 4mL of 50% slurry) containing 0.02% sodium azide Binding Capacity: ~10-20mg human IgG/mL of resin Storage: Upon receipt store product at 4-8°C. Product is shipped at ambient temperature. Table of Contents Introduction ................................................................................................................................................................................. 1 Important Product Information .................................................................................................................................................... 1 Gravity-flow Column Procedure for Antibody Purification Using Protein L Agarose ................................................................ 2 A. Additional Materials Required ...................................................................................................................................... 2 B. Immunoglobulin Purification Procedure ....................................................................................................................... 2 Troubleshooting ........................................................................................................................................................................... 3 Additional Information ...............................................................................................................................................................
    [Show full text]