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, telephone biology, and immunology, they describe step by step equipment designed to query how MHC-binding peptides are generated, to check how peptides are dropped at MHC molecules, to investigate MHC peptide-binding styles, and to assay the T-cell reaction to the MHC/peptide advanced. Emphasis is given these technical steps serious for experimental luck which are usually 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 sensible instruments that would expand the questions that may be requested, and successfully be responded, touching on antigen processing/presentation.
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Extra info for Antigen Processing and Presentation Protocols (Methods in Molecular Biology Vol 156)
11. 12. 174, respectively) for comparison, rather than an unrelated cell type. 174 express the TAP molecules that RMA-S and T2 lack, but they also express additional proteins. To definitively state that the lack of TAP accounts for a lack of presentation of exogenous Ag, TAP must be transfected, or otherwise expressed, in cells originally lacking it, and a restoration of presentation demonstrated. For contamination to be ruled out, it is essential that the peptide representing the minimal T-cell determinant is known, to allow direct comparison between intact and peptide Ag.
3. Anticlass I mAbs for use in cytofluorographic analysis. They are available from different manufacturers, unlabeled or conjugated to fluorescein isothiocyanate (FITC), as well as other fluorophores. 4. If the anticlass I Ab is not directly labeled, a FITC-conjugated anti-immunoglobulin Ab, specific for the anticlass I Ab. 5. Ethidium homodimer (Molecular Probes, Eugene, OR). Stock solution in PBS–BSA (100 µg/mL). 6. Flow cytometer. 7. 3. 6. 1. Infection of Target Cells with VV 1. 3. 2. rVV viruses expressing the proteins of interest.
This is also relevant when UbMNP is used as a control, because some co-translational degradation of the protein is observed, presumably before removal of the ubiquitin moiety, whereas the final product is stable. Such an effect is not observed, however, with the wild type NP. 5. Often, the lysate is too viscous to handle easily. This can be solved by shearing the DNA with a probe sonicator, passing through a 23 G needle, or keeping the lysates overnight at 4°C. 28 Antón, Bennink, and Yewdell 6.
Antigen Processing and Presentation Protocols (Methods in Molecular Biology Vol 156) by Solheim J.C.