Solheim J.C.'s Antigen Processing and Presentation Protocols (Methods in PDF

By Solheim J.C.

Well-recognized and cutting edge experimentalists element their state-of-the-art tools for learning the antigen processing and presentation. Drawing on services from biochemistry, mobilephone biology, and immunology, they describe step by step tools designed to question how MHC-binding peptides are generated, to check how peptides are dropped at MHC molecules, to research MHC peptide-binding styles, and to assay the T-cell reaction to the MHC/peptide advanced. Emphasis is given these technical steps severe for experimental good fortune which are frequently passed over from tools released within the basic literature. Eminently obtainable and cutting-edge, Antigen Processing and Presentation Protocols offers either new and skilled investigators with hugely useful instruments that may expand the questions that may be requested, and successfully be responded, pertaining to antigen processing/presentation.

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Extra info for Antigen Processing and Presentation Protocols (Methods in Molecular Biology Vol 156)

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2. 5, containing 1% BSA (use 100 µL glycine buffer/2 × 106 cells). 3. Incubate for 3 min at 37°C, and neutralize immediately with a large excess of medium. 4. Pellet cells, and resuspend in culture medium containing the appropriate inhibitor. A control with BFA (5 µg/mL), a drug that blocks transport from the ER to the Golgi, and thus blocks class I cell surface expression should be included. 5. Incubate for 5–8 h at 37°C, rotating. 6. Pellet cells. 02% NaN3, to prevent internalization of cell surface molecules (see Note 7).

1. Inhibitor Stocks Table 1 shows a list of commercially available proteasome inhibitors commonly used in studies of Ag presentation, solvents, and concentrations of stock solutions. These are carbobenzoxy-leucyl-leucyl-leucinal (zLLL, also known as MG132); N-acetyl-leucyl-leucyl-norleucinal (AcLLnL, calpain inhibitor I); carbobenzoxy-leucyl-leucyl-norvalinal (zLLnV, MG115); AcLLM, calpain Proteasome Inhibitors 21 inhibitor II, a control inhibitor which does not affect the proteasome; lactacystin; CLβL, the active component of lactacystin; and 4-hydroxy-5-iodo3-nitrophenylacetyl-leucyl-leucyl-leucyl-vinylsulfone (NLVS).

The lacZ hybridomas have the added advantage that activation can be assessed on a single-cell basis, allowing recognition of rare events within an APC population (8). 2. Strategies Once a suitable readout system has been established, one may proceed with investigating the trafficking of Ag and MHC-I molecules. Initially, it is imperative to rule out peptide contamination in the Ag preparation. Failure to do so will leave the possibility that peptide contaminants in the Ag preparation are binding to MHC-I directly, either at the cell surface or at another site.

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