Dimer/Polymer Affinity Measurement Service

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Creative Biolabs provide label-free and high-throughput Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI) technologies based services, which is capable of satisfying any of your specific demand. </p> <h2 class="h2_tit page_tab_margin" id="tab1"> Background </h2> <p> Monomer is the simplest form of a carbohydrate, nucleic acid, lipid, or protein (such as glucose, nucleotide and amino acid). Dimer is a pairing of structurally similiar monomers (such as sucrose and phospholipid). Polymer is a set of many indentical monomers associated together through polymerization (such as dynamic actin filaments and microtubules). Dimers or polymers are joined by either covalent bonds or hydrogen bonds, while we focus on the affinity analysis of the latter form. Y. R. Kim <em>et al</em>. performed a process named enzymatic surface initiated polymerization (ESIP) with SPR system to work out affinity analysis for polyhydroxybutyrate (PHB) (Figure 1). ESIP has been extensively studied due to its unique ability which allows the robust and dense polymers to grow directly from the gold surface using an immobilized initiator. </p> <p class="show-center"> <img src="static/img/Affinity-Measurement-Services-for-Dimers-Polymers-1.png" alt="Figure 1. Different concentration of PHA synthase binding to the mixed SAMs and subsequent polymerization (left panel). SPR sensorgram showing the binding of His-tag PHA synthase to a Ni-NTA functionalized gold surface and the subsequent dissociation of the same protein from the surface by free imidazole (right panel). PHA, polyhydroxyalkanoate; SAMs, Self-assembled mono- layers. (Macromol. Biosci., 2006)" width="500" height="186" loading="lazy" />Figure 1. Different concentration of PHA synthase binding to the mixed SAMs and subsequent polymerization (left panel). SPR sensorgram showing the binding of His-tag PHA synthase to a Ni-NTA functionalized gold surface and the subsequent dissociation of the same protein from the surface by free imidazole (right panel). PHA, polyhydroxyalkanoate; SAMs, Self-assembled mono- layers. (Macromol. Biosci., 2006) </p> <p> Take XMAP215 polymerase as another example, which regulates microtubule growth via its repeat domains locate at the N termini (TOG domains), which binds tubulin dimers, therefore the affinity for tubulin can be observed through the polymerization kinetics. </p> <p class="show-center"> <img src="static/img/Affinity-Measurement-Services-for-Dimers-Polymers-2.png" alt="Figure 2. Model of TOG12+++ on the plus end of a microtubule. TOG12+++, the TOG12 fragment with the strong microtubule-binding domain. (PNAS, 2011)" width="400" height="202" loading="lazy" />Figure 2. Model of TOG12+++ on the plus end of a microtubule. TOG12+++, the TOG12 fragment with the strong microtubule-binding domain. (PNAS, 2011) </p> <p> As shown in Fiugre 2, the following reaction scheme of the microtubule polymerization is: </p> <p class="show-center"> <img src="static/img/Affinity-Measurement-Services-for-Dimers-Polymers.png" alt="" width="430" height="125" loading="lazy" /> </p> <p> Where microtubule end (Tn) represents the enzyme, free tubulin dimer (T) is the substrate, XMAP215 (X) is the activator. The relative affinity of the activator for the intermediate state is given by α, while the association rate change of the intermediate state due to the activator is given by α', thus, α and α' are defined as : </p> <p class="show-center"> <img src="static/img/Affinity-Measurement-Services-for-Dimers-Polymers-3.png" width="450" height="54" loading="lazy" /> </p> <p> From the formula above, we can confirm the tubulin-binding affinity correlates with the activity of XMAP215, which localizes to the growing microtubule ends primarily through microtubule lattice diffusion. Finally, the flux of XMAP215 molecules to the microtubule end is given by: </p> <p class="show-center"> <img src="static/img/Affinity-Measurement-Services-for-Dimers-Polymers-4.png" width="200" height="102" loading="lazy" /> </p> <p> Where D indicates the diffusion coefficient of XMAP215 on the microtubule lattice, Kon and Koff are the association and dissociation rate constants, respectively, for the binding of XMAP215 to microtubule. From the above formula, we notice that the activity of XMAP215 polymerase increases with the microtubule-binding affinity. </p> <p class="show-center"> <img src="static/img/Affinity-Measurement-Services-for-Dimers-Polymers-5.png" alt="Figure 3. The affinity analysis of XMAP215 polymerization " width="400" height="154" loading="lazy" /> Figure 3. The affinity analysis of XMAP215 polymerization </p> <p> Mutation or removal in XMAP215 leads to a higher KD of the polymerization without the change of νmax. Once XMAP215 targets to the microtubule end, the tubulin affinity can determine the maximal growth rate (νmax) at any fixed tubulin concentration (Figure 3, A). The graph shows theoretical dose response of a protein with a constant νmax and an altered Kon during microtubule-lattice binding: 4 x reduced in red, 2 x reduced in green, not reduced in blue. We can note that the KD value is changed (Figure 3, B). </p> <h2 class="h2_tit page_tab_margin" id="tab2"> Affinity Measurement for Dimers/Polymers </h2> <p> Creative Biolabs can provide custom affinity and kinetics measurements for dimers and polymers based on label-free and high-throughput SPR and BLI technologies. All the data analysis will be performed and documented. Please feel free to contact us for a detailed quote. </p> <p> Other optional <a href="/Antibody-Affinity-Measurement.html">Antibody Affinity Measurement</a> Services: </p> <ul class="ullist"> <li> <a href="/Cell-Line-Affinity-Measurement.html">Cell Line Affinity Measurement</a> </li> <li> <a href="/Affinity-Measurement-Services-for-Specific-Antibodies.html">Affinity Measurement Services for Specific Antibodies</a> </li> <li> <a href="/Membrane-Proteins-Affinity-Measurement.html">Membrane Protein Affinity Measurement</a> </li> <li> <a href="/Affinity-Measurement-Services-for-Bivalent-Antigens.html">Affinity Measurement Services for Bivalent Antigens</a> </li> <li> <a href="/Affinity-Measurement-Services-for-Small-Molecules.html">Affinity Measurement Services for Small Molecules</a> </li> <li> <a href="/Affinity-Measurement-Services-for-Cell-Lysates.html">Affinity Measurement Services for Cell Lysates</a> </li> <li> <a href="/Affinity-Measurement-Services-for-Various-Ligands-and-Analytes.htm">Affinity Measurement Services for Various Ligands and Analytes</a> </li> <li> <a href="/Biacore-System.html">Biacore System</a> </li> <li> <a href="/ProteOn-System.html">ProteOn System</a> </li> <li> <a href="/Octet-system.html">Octet system</a> </li> </ul> <div class="reference"> <strong>References</strong> <ol class="ollist decimal"> <li> M. Vidal, et al. (2004). Design of peptoid analogue dimers and measure of their affinity for Grb2 SH3 domains. <em>Biochemistry</em>. 43 (23): 7336-7344 </li> <li> P. O. Widlund, et al. (2011). XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region. <em>PNAS</em>. 108 (7): 2754-2746. </li> <li> S. Elbaum-Garfinkle et al. (2014). Tau mutants bind tubulin heterodimers with enhanced affinity. <em>PNAS</em>. 111 (17): 6311-6316. </li> <li> Y. R. Kim, et al. (2006). Real-Time Analysis of Enzymatic Surface-Initiated Polymerization Using Surface Plasmon Resonance (SPR). 6 (2): 145-152. </li> </ol> </div> <script type="text/javascript"> window.addEventListener('load',function(){ faqlist(4); }); </script> <h2 class="h2_tit page_tab_margin" id="tab3"> FAQ </h2> <ol class="ollist faqlist"> <li> <span class="sFAQTit">How do label-free SPR and BLI technologies measure the affinity of dimers or polymers?</span> <p> Label-free technologies like Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI) measure the affinity of dimers or polymers by monitoring real-time binding interactions without the use of fluorescent, radioactive, or enzymatic labels. SPR measures changes in the refractive index near a sensor surface to which one partner of the binding pair is immobilized, while BLI uses an optical fiber tip coated with a biocompatible matrix that captures one binding partner. The interaction of the dimer or polymer with its partner causes a shift in the interference pattern of light, which is directly correlated to the binding affinity and provides a quantitative measure of the interaction strength. </p> </li> <li> <span class="sFAQTit">What advantages do high-throughput SPR and BLI offer in studying the kinetics of polymers or dimers?</span> <p> These technologies allow for rapid and simultaneous analysis of multiple samples under various conditions, which is critical for effective screening of binding behaviors and optimization processes. Both SPR and BLI provide detailed kinetic data including association rates (kon) and dissociation rates (koff), enabling researchers to derive the affinity constants (KD) of interactions. This capability is invaluable in drug development and molecular interaction studies where understanding the binding dynamics is essential for characterizing the biological functions of molecules. </p> </li> <li> <span class="sFAQTit">Can SPR and BLI technologies differentiate between different types of dimer or polymer interactions?</span> <p> Both SPR and BLI technologies are capable of differentiating between different types of interactions among dimers or polymers. These technologies measure the specific binding events in real-time, allowing researchers to observe the behavior of complex formations, including homo- or heterodimerization and higher-order polymerizations. By analyzing the binding curves and comparing them against known interaction models, it is possible to discern whether the binding involves simple one-to-one interactions, cooperative binding, or competitive mechanisms. </p> </li> <li> <span class="sFAQTit">How do SPR and BLI handle the challenge of measuring high-affinity interactions of polymers and dimers?</span> <p> Measuring high-affinity interactions using SPR and BLI can be challenging due to rapid on-rates and extremely slow off-rates, which may lead to saturation of the sensor surface and prolonged dissociation times. To address these challenges, both technologies employ strategies like using low analyte concentrations, enhancing fluidics systems for better washout of bound molecules, and optimizing the regeneration conditions to efficiently dissociate the bound complexes without damaging the sensor surface. Additionally, advanced data analysis techniques are used to accurately calculate the kinetic constants from the obtained binding curves, ensuring reliable measurement of high-affinity interactions. </p> </li> <li> <span class="sFAQTit">How do SPR and BLI compare in terms of sensitivity and specificity when measuring interactions of dimers or polymers?</span> <p> SPR and BLI both offer high sensitivity and specificity, but their performance can vary based on the specific application. SPR is typically noted for its high sensitivity due to its ability to detect small changes in refractive index caused by molecule binding on the sensor surface. BLI, although slightly less sensitive than SPR in some cases, provides the advantage of direct measurement of the interference pattern without the need for surface regeneration, making it particularly useful for capturing transient or weak interactions. Both technologies are highly specific, capable of distinguishing specific binding events from nonspecific bindings by careful control of experimental conditions and proper reference channel usage. </p> </li> <li> <span class="sFAQTit">What are the limitations of using SPR and BLI for the study of large polymeric structures?</span> <p> The size and complexity of these molecules can lead to steric hindrances and mass transport limitations, affecting the accuracy of binding measurements. Large polymers can also cause signal artifacts due to their extended structures overlapping with multiple sensor areas. To mitigate these issues, it's crucial to optimize the immobilization strategies, use dilute solutions to minimize mass transport problems, and carefully interpret the sensorgrams to distinguish between true binding events and artifacts. </p> </li> </ol> <h2 class="h2_tit page_tab_margin" id="tab4"> Resources </h2> <p> Use the resources in our library to help you understand your options and make critical decisions for your study. </p> <div class="tab_nav"> <span class="tab_choosebg">Videos</span><span>Podcasts</span><span>Infographic</span><span>Flyer</span><span>Case study</span><span>Articles</span> </div>  <div class="tab_cont" style="display:block;"> <div class="row pagerow_pt"> <div class="col-md-4"> <div class="pagerow_pt_unit"> <img loading="lazy" src="../static/img/introduction-of-recombinant-antibody.jpg" alt="" /> </div> <a href="https://youtu.be/0-yZ9dTGg18" target="_blank">Introduction of Recombinant Antibody</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit"> <img loading="lazy" src="../static/img/immunogenicity-overview-of-therapeutic-biologics.jpg" alt="" /> </div> <a href="https://youtu.be/J430752_dTo" target="_blank">Immunogenicity overview of therapeutic biologics</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit"> <img loading="lazy" src="../static/img/video-development-of-monoclonal-antibodies-s.png" alt="" /> </div> <a href="https://youtu.be/NGqipRkxXiY" target="_blank">Antibody Library Screening - Creative Biolabs</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit"> <img loading="lazy" src="../static/img/antibody-based-cancer-therapies-creative-biolabs.jpg" alt="" /> </div> <a href="https://youtu.be/_Cqj5gphNug" target="_blank">Antibody based Cancer Therapies - Creative Biolabs</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit"> <img loading="lazy" src="../static/img/video-de-novo-antibody-sequencing-service.jpg" alt="" /> </div> <a href="https://youtu.be/lcdGTNKbaGQ" target="_blank"><em>De Novo</em> Antibody Sequencing Service</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit"> <img loading="lazy" src="../static/img/video-100-identification-of-leucine-and-isoleucine-in-de-novo-antibody-sequencing.jpg" alt="" /> </div> <a href="https://youtu.be/lcdGTNKbaGQ" target="_blank">100% Identification of Leucine and Isoleucine in <em>De Novo</em> Antibody Sequencing</a> </div> </div> </div>  <div class="tab_cont"> <div class="therapy_ad"> <a href="therapeutic-antibody.html"> <img loading="lazy" src="../static/img/podcasts-ad.png" width="1700" height="500" srcset="../static/img/podcasts-ad.webp 1x,../static/img/podcasts-ad.webp 2x,../static/img/podcasts-ad.webp 3x" alt="In this brand new podcast series, we will show you everything you want to know about CAR-T therapy, including the mechanism, current applications, technology limitations, and potential strategies." /></a> </div> </div>  <div class="tab_cont"> <div class="row pagerow_pt"> <div class="col-md-4"> <div class="pagerow_pt_unit_2"> <img loading="lazy" src="/resource/images/A-Brief-Introduction-to-Antibodies-1.jpg" width="600" height="500" alt="" /> </div> <a href="https://go.creative-biolabs.com/a-brief-introduction-to-antibodies.html">A Brief Introduction to Antibodies</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit_2"> <img loading="lazy" src="/resource/images/Antibody-Library-Screening-Creative-Biolabs.jpg" width="600" height="500" alt="" /> </div> <a href="https://go.creative-biolabs.com/antibody-library-screening.html ">Antibody Library Screening</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit_2"> <img loading="lazy" src="/resource/images/De-Novo-Antibody-Seuqencing-Service-Creative-Biolabs.jpg" width="600" height="500" alt="" /> </div> <a href="https://go.creative-biolabs.com/de-novo-antibody-sequencing-service.html"><em>De Novo</em> Antibody Sequencing Service</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit_2"> <img loading="lazy" src="/resource/images/Antibody-Based-Therapies-1.jpg" width="600" height="500" alt="" /> </div> <a href="https://go.creative-biolabs.com/antibody-based-therapies.html">Antibody-Based Therapies</a> </div> </div> </div>  <div class="tab_cont"> <div class="row pagerow_pt"> <div class="col-md-4"> <div class="pagerow_pt_unit_2"> <img loading="lazy" src="/resource/images/Magic-Therapeutic-Antibody-Discovery-Platform-Flyer-Creative-Biolabs.jpg" width="600" height="500" alt="" /> </div> <a href="/resource/pdf/com/flyers/Magic-Therapeutic-Antibody-Discovery-Platform-Flyer-Creative-Biolabs.pdf">Magic<sup>TM</sup> Therapeutic Antibody Discovery Platform</a> </div> </div> </div>  <div class="tab_cont"> <div class="row pagerow_pt"> <div class="col-md-4"> <div class="pagerow_pt_unit_2"> <img loading="lazy" src="/resource/images/Antibody-Development-Case-Study-com.jpg" width="530" height="500" alt="" /> </div> <a href="https://go.creative-biolabs.com/antibody-development-case-study.html">Antibody Development Case Study</a> </div> </div> </div>  <div class="tab_cont"> <div class="row pagerow_pt"> <div class="col-md-4"> <div class="pagerow_pt_unit"> <img loading="lazy" src="../static/img/what-is-antibody-de-novo-sequencing-1.jpg" width="600" height="500" alt="" /> </div> <a href="/what-is-antibody-de-novo-sequencing.html">What Is Antibody <em>De Novo</em> Sequencing?</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit"> <img loading="lazy" src="../static/img/what-is-protein-mass-spectrometry-technology-1.jpg" width="600" height="500" alt="" /> </div> <a href="/what-is-protein-mass-spectrometry-technology.html">What Is Protein Mass Spectrometry Technology?</a> </div> <div class="col-md-4"> <div class="pagerow_pt_unit"> <img loading="lazy" src="../static/img/hybridoma-sequencing-technology-assisting-the-r-d-and-production-1.jpg" width="600" height="500" alt="" /> </div> <a href="/hybridoma-sequencing-technology-assisting-with-the-r-d-and-production-of-monoclonal-antibodies.html">Hybridoma Sequencing Technology - 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