Download Antigen Processing and Presentation Protocols by Jill R. Beyette, Timothy Hubbell (auth.), Joyce C. Solheim PDF

By Jill R. Beyette, Timothy Hubbell (auth.), Joyce C. Solheim (eds.)

The processing and presentation of antigens by means of significant histocompatibility advanced (MHC) molecules is an important immunological phenomenon in infectious ailment, malignancy, autoimmune sickness, and transplantation. In Antigen Processing and Presentation Protocols, well-recognized and cutting edge experimentalists element their state of the art equipment for learning this advanced approach. Drawing on services from biochemistry, mobile biology, and immunology, they describe step by step tools designed to query how MHC-binding peptides are generated, to check how peptides are brought to MHC molecules, to investigate MHC peptide-binding styles, and to assay the T phone reaction to the MHC/peptide advanced. every one procedure is gifted in adequate aspect to be with no trouble reproducible and comprises notes approximately strength pitfalls and easy methods to keep away from mess ups. Emphasis is given these technical steps severe for experimental good fortune which are usually passed over from equipment released within the fundamental literature.
Eminently available and state of the art, Antigen Processing and Presentation Protocols presents either new and skilled investigators with hugely sensible instruments that may expand the questions that may be requested, and successfully be replied, relating antigen processing/presentation.

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2. 3. Dissolve FITC slowly into DMSO, to give a final concentration of 1 mg/mL. 4. Add a 20-fold molar excess of FITC slowly to the Ag preparation, in 5 μL aliquots, while stirring in an ice bath. 5. Allow the conjugation reaction to proceed in the dark for 8 h at 4°C, while stirring continually. 6. Bring the Ag preparation to RT and add NH 4 Cl to a final concentration of 50 mM. Allow the reaction to quench for 2 h at RT. 7. 4, by running PBS through it. 8. Pass the Ag conjugate through the Biogel P-10 column, and collect the conjugated Ag, free of unconjugated FITC.

Estimate the amount of radioactivity incorporated in the different samples by spotting 5 μL of each sample, in triplicate, on glass-fiber filters. Wash filters with 10% TFA (w/v), dry, and place in appropriate vials with biodegradable scintillation fluid, and count the filters in a β-counter. 8. Adjust the volumes of each sample used for immunoprecipitation, so that each sample contains the same amount of incorporated 35S-Met. Prepare two aliquots with each sample. 9. To one of the aliquots, add a class I-binding synthetic peptide (final concentration 5 μg/mL).

J. C. (1997) Regulation of class I-restricted epitope processing by local or distal flanking sequence. J. Immunol. 158, 1727–1733. , Antón, L. , Snyder, H. , Bennink, J. , and Yewdell, J. W. Generation of MHC class I-associated peptides is only partially inhibited by proteasome inhibitors. Involvement of nonproteasomal proteases in antigen processing? J. Immunol. 159, 554–564. , and Townsend, A. (1997) Proteasome-specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells.

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