Get Protein Arrays: Methods and Protocols PDF

By Hendrik Weiner, Thomas Faupel, Konrad Büssow (auth.), Eric T. Fung MD, PhD (eds.)

ISBN-10: 158829255X

ISBN-13: 9781588292551

Protein arrays make attainable the detection and quantitation of many proteins at the same time, hence permitting researchers to invite primary questions on organic procedures and to find biomarkers that may be used diagnostically. In Protein Arrays: tools and Protocols, cutting edge experimentalists describe intimately the equipment they've got constructed to synthesize and build protein arrays for easy and medical study. The authors current protocols to create and immobilize the trap substrate-the first job in designing a protein array-using numerous affinity seize reagents, together with antibodies, peptides, aptamers, biotin, chemical reagents, and chromatographic substrates. as soon as synthesized, those arrays can be utilized to research protein-protein interactions and posttranslational ameliorations, corresponding to phosphorylation, in addition to to find and represent power diagnostic markers. Protocols to complete those projects also are offered. The protocols provided stick to the winning equipment in Molecular Biology™ sequence structure, each supplying step by step laboratory directions, an creation outlining the main in the back of the process, lists of kit and reagents, and pointers on troubleshooting and fending off identified pitfalls.
different and hugely functional, Protein Arrays: equipment and Protocols bargains easy and scientific investigators a extensive spectrum of methods to the iteration of protein arrays, in addition to their makes use of in biomarkers discovery, in assay improvement, in scientific pattern trying out, in sign transduction research and characterization, and for growing the subsequent iteration of molecular tools.

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Additional resources for Protein Arrays: Methods and Protocols

Example text

4. 2. Prepare the detection antibody solution. a. Mix the appropriate amounts of Cy3-labeled and Cy5-labeled antihapten antibodies, based on the solution volume per array times the concentration of each antibody times the number of arrays. The optimal concentration of the antibodies can be determined by titration (often around 10 µg/mL). b. 4. c. Add 10X blocker. Use 1/10 of the intended final volume of the antibody mix. d. Add 1X PBS to the final volume. 3. ) on the arrays, and wash and dry the arrays according to steps 1–7 above.

3. Using a red-hot syringe needle, make three deep holes on the membrane perimeter to introduce a coordinate system. Label the membrane. 4. Block the membrane in 1% BSA for 1 h at room temperature with gentle rocking in a 150-mm cell culture Petri dish. 5. Mix 5 µg of the biotinylated protein target with STRAP at a molar ratio of 4:1 in 100 µL of TBST. Incubate 10–15 min at room temperature (see Note 7). 6. 5% of BSA. 7. Incubate the blocked membrane in 30 mL of target–STRAP solution at 4°C overnight with gentle rocking.

At lower plaque densities. 7. Use a target-to-STRAP ratio of 4:1 or higher for multivalent target presentation if detection of weak interactions and low stringency conditions are desired, and the respective ratio of 1:4 or lower in case of strong interactions and high-stringency conditions. E. Bredesen). References 1. Kay, B. , and McCafferty, J. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual. Academic, San Diego, CA. 2. Cabilly, S. (1998) Methods in Molecular Biology, vol. 87: Combinatorial Peptide Library Protocols.

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Protein Arrays: Methods and Protocols by Hendrik Weiner, Thomas Faupel, Konrad Büssow (auth.), Eric T. Fung MD, PhD (eds.)

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