Manual Avidin-Biotin Interactions: Methods and Applications

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Omar Olmos-Lopez and Ms. Lola R. Justin Shearer, Andrea R. Gomez-Fernandez and Dr. Roberto F. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Introduction The extremely high affinity of biotin B 7 , vitamin H for avidin AV and streptavidin SAV is widely exploited in biotechnology and biochemistry in a vast array of applications [ 1 , 2 ]. Download: PPT. Experimental procedures Materials Probes and solution conditions.

Protein and active site concentrations. Association stopped-flow kinetics. Dissociation reactions of dye-labeled biotin complexes. Spectroscopic properties. Methodologies The following experiments were carried out by at least six times, unless indicated, and the reported errors correspond to the standard deviation.

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Steady-state anisotropy r ss. Quantum yields QY. Dissociation reactions of the complexes. Time-resolved anisotropy. Results and discussion Active avidin binding sites The avidin and streptavidin proteins are tetramers in solution. Association rate constants k on of biotin binding to avidin Dye-labeled biotin association rate constants by stopped-flow methodology. Fig 4. Fig 5. Table 1. Comparison of the AV-BcO association rate constants k on obtained by fluorescence change, , and corrected fluorescence anisotropy,.

Immunochemical Applications of Avidin-Biotin Technology | Springer Nature Experiments

Table 2. Temperature dependent association rate constants k on and thermodynamic values of the dye-labeled B 7 binding to AV and SAV. Unlabeled biotin association rate constants by relaxation kinetics methodology. Fig 6. Non-cooperative biotin binding to avidin sites. Comparisons with other AV-B kinetic studies. Effect of AV glycosylation on the biotin binding kinetics. Association reaction of unlabeled and dye-labeled biotin binding to streptavidin Dye-labeled biotin association reactions to SAV. Comparisons with other SAV-B 7 association kinetic studies.

Fig 7. Comparisons with other biotinylated DNA kinetic studies. Association reactions of biotin vs. Fig 9. Fig Table 3. Table 4. Supporting information. S1 Fig. S2 Fig. S3 Fig. S1 Table. Lifetimes of dye-labeled B 7 probes and protein complexes. S1 File. Excel file with data values. Acknowledgments Roberto F Delgadillo thanks Dr. References 1. Wilchek M, Bayer EA. Introduction to avidin-biotin technology. Methods Enzymol. View Article Google Scholar 2. Wilchek M. My life with affinity.

The avidin-biotin system

Protein Sci. View Article Google Scholar 3. Bayer EA, Wilchek M. Methods Biochem Anal. The biotin- strept avidin system: principles and applications in biotechnology. Clin Chem. The avidin-biotin complex in immunology. Immunol Today. Biomed Microdevices. View Article Google Scholar 7. Ultrastable artificial binding pairs as a supramolecular latching system: A next generation chemical tool for proteomics. Acc Chem Res. Anal Chem. Quantum dots for live cells, in vivo imaging, and diagnostics.

Howarth M, Ting AY. Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin. Nat Protoc. Jain A, Cheng K. The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis.

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View Article Google Scholar Homogeneous time resolved fluorescence resonance energy transfer using rare earth cryptates as a tool for probing molecular interactions in biology. Cisbio Appl Note. Int J Mol Sci. DNA condensation by high-affinity interaction with avidin. J Mol Recognit. Transgenic avidin maize is resistant to storage insect pests. Nat Biotechnol. Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family. Biochem J. J Mol Biol. Tamavidins—Novel avidin-like biotin-binding proteins from the Tamogitake mushroom. FEBS J. Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis.

BMC Struct Biol. Bradavidin II from Bradyrhizobium japonicum: A new avidin-like biotin-binding protein. Biochim Biophys Acta—Proteins Proteomics. Novel avidin-like protein from a root nodule symbiotic bacterium, Bradyrhizobium japonicum. J Biol Chem. Eur J Biochem. Avidin related protein 2 shows unique structural and functional features among the avidin protein family.

BMC Biotechnol. Factors Dictating the Pseudocatalytic Efficiency of Avidins. Chicken avidin exhibits pseudo-catalytic properties. Biochemical, structural, and electrostatic consequences. Critical importance of loop conformation to avidin-enhanced hydrolysis of an active biotin ester. Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family.

BMC Genomics. Three-dimensional structures of avidin and the avidin-biotin complex. Proc Natl Acad Sci. Rational design of an active avidin monomer. Green NM. The use of [14C]biotin for kinetic studies and for assay. Biotin-avidin binding kinetics measured by single-molecule imaging. High-throughput DNA droplet assays using picoliter reactor volumes. Monitoring of real-time streptavidin-biotin binding kinetics using droplet microfluidics.

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  • Cooperative hydrogen bond interactions in the streptavidin-biotin system. Spectrophotometric Determination of Avidin and Biotin. FEBS Lett. J Comput Aided Mol Des. TATA-binding protein recognition and bending of a consensus promoter are protein species dependent. PLoS One.

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    Spectroscopic properties of fluorescein and rhodamine dyes attached to DNA. Photochem Photobiol. Giblin DE. A modular instrument for the measurement of transient circular dichroism, fluorescence polarization and emission anisotropy. University of Nebraska-Lincoln; Correction of fluorescence spectra and measurement of fluorescence quantum efficiency. Measurement of photoluminescence quantum yields. J Phys Chem. Mueser TC. Steady state fluorescence and fluorescence anisotropy studies of ligand binding and kinetics.

    Foss SD. Resolution of multiphasic reactions by the combination of fluorescence total-intensity and anisotropy stopped-flow kinetic experiments. Biophys J. Bucci E, Steiner RF. Anisotropy decay of fluorescence as an experimental approach to protein dynamics. Biophys Chem. Cantor C. San Francisco. Freeman and Company; Time-resolved fluorescence resonance energy transfer studies of DNA bending in double-stranded oligonucleotides and in DNA-protein complexes.

    Lipari G, Szabo A. Effect of librational motion on fluorescence depolarization and nuclear magnetic resonance relaxation in macromolecules and membranes. Sinha B. The synthesis and use of spin-labeled analogs of biotin in the study of avidin. Biotin-binding Proteins: Overview and Prospects. Influence of globin structure on the state of the heme. Human deoxyhemoglobin. Biotin binding to avidin. Oligosaccharide side chain not required for ligand association. John W. Moore RGP. Kinetics and Mechanism. Wiley Interscience; Piran U, Riordan WJ.

    Dissociation rate constant of the biotin-streptavidin complex. J Immunol Methods. Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotinfluorescein. J Phys Chem B. Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: A multifunctional quantitative assay for biotin.

    Anal Biochem. Single Mol. Connors KA. Extremely high thermal stability of streptavidin and avidin upon biotin binding. Biomol Eng.

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    Conformational flexibility of avidin: The influence of biotin binding. Biochem Biophys Res Commun. Green N. Thermodynamics of the binding of biotin and some analogues by avidin. Stjurkuusk J, Wadso I. Swamy MJ. Thermodynamic analysis of biotin binding to avidin. A high sensitivity titration calorimetric study. Biochem Mol Biol Int. A simple spectrophotometric streptavidin-biotin binding assay utilizing biotinfluorescein.

    J Biochem Biophys Methods. Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex. Streptavidin-biotin binding energetics. Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system. J Am Chem Soc. Weber P. Acta Cryst. Absorption and fluorescence properties of fluorescein. Spectrochim Acta Part A.

    Characterization of fluorescein-oligonucleotide conjugates and measurement of local electrostatic potential. Doktorsavhandlingar vid Chalmers Tek Hogsk. Parkhurst LJ. This is because of the nature of the indirect interactions, nonspecific binding and antibody contamination that may mask detection. It is therefore important to conduct parallel negative controls, deliberately add an excess of the antigen, or covalently crosslink the proteins before immunoprecipitation.

    Membrane-permeable cross-linking reagents are used in vivo. The excess amino groups can inhibit cross-linking. In addition, the cross-linked protein can be released from the immune complex by cleavage of the crosslink with DTT. After the cross-linking, immunoprecipitation of the ligand protein is performed.

    Peptides or proteins can be labeled before cross-linking. This method will give a clean result. LifeTein routinely synthesizes biotinylated peptides for use in protein-protein interaction studies. Although biotin can be introduced either N- or C-terminally via lysine residues , we recommend the use of an N-terminal modification owing to its low cost, higher success rate, shorter turnaround time, and ease of operation.

    Peptides are synthesized from the C-terminus to the N-terminus; therefore, the N-terminal modification is the last step in the SPPS protocol, and no additional specific coupling steps are required. In contrast, a C-terminal modification requires additional steps, and is usually more complex. However, in principle the biotin can be positioned anywhere. Biotin can be separated from the peptide by a variety of different linkers or spacers.

    Nevertheless, it is recommended that a flexible spacer such as Ahx a 6-carbon linker is included to render the biotin label more stable or flexible. Biotinylated peptides can also be immobilized tightly on streptavidin columns. Nevertheless, an N-terminal amine or an amine group on a lysine residue is needed for successful binding reactions. Home Services. Protein-Protein Interactions: Methods for Detection and Analysis There are a large number of protein-protein interactions in the cell. Pull-down Introduction Applications 1. Protein affinity chromatography: The most straightforward method is to use the peptide as bait in affinity pull-down experiments and followed by detection of binding proteins.

    The sepharoses are incubated with a sample of interest. To detect weak protein-protein interactions, the concentration of bound protein should be as high as possible. The amount of extract applied to the column is very critical. If too little extract is used, there will be too small protein retained to be detecte. The affinity chromatography is incredibly sensitive and can be used to test all proteins in an extract equally. It is easy to make different peptides at its critical residues for specific interactions. The mutant derivatives of peptides can be used to examine the crucial domain of a protein.

    Most unbound proteins pass through the columns. A control column should be used to avoid a false-positive result because the protein may bind to another protein by ionic charge interactions. The protocol used to Pull-Down Biotin-labeled Peptides. However, during the blotting procedure, proteins are recovered by removing denaturants. Another option is to fraction the proteins by a nondenaturing gel system to retain the biological activity. The protein of interest can then be radioactively labeled, or biotinylated to probe gel strip or a nitrocellulose membrane after gel transfer.

    This assay is not quantitative. Immunoprecipitation One of the most commonly used methods for verification of protein-protein interactions is Co-immunoprecipitation Co-IP. Bait complexes are captured from cell lysate using a specific antibody. It is better to use monoclonal antibody for the precipitation. This is to confirm that the contaminating antibody does not precipitate the coprecipitated protein.

    Polyclonal antibodies are usually preadsorbed against extracts to remove contaminants. The complex is then immobilized to protein A or protein G sepharose beads. After washing steps, the antibody, the bait, and proteins interacted with bait are eluted from the beads. The bound proteins can then be identified by Mass Spec or by immunoblotting.

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    Co-IP Optimization Use non-denaturing lysis buffers with low ionic strength i. The samples should be handled gently in order not to disrupt the bound target complex. Wash the immune complexes thoroughly to avoid high background.