Antigen Processing: Methods and Protocols by Stefan Tenzer, Tobias Hain, Hendrik Berger, Hansjörg Schild

By Stefan Tenzer, Tobias Hain, Hendrik Berger, Hansjörg Schild (auth.), Peter van Endert (eds.)

Antigen processing is a organic approach that prepares antigens for the presentation to important cells within the immune approach referred to as T lymphocytes. In Antigen Processing: tools and Protocols, professional researchers within the box offer a entire set of protocols for learning presentation of antigens produced within the normal processing pathways for MHC classification I and sophistication II molecules. The chapters stick with chronology of intracellular processing occasions, finishing with acceptance of peptide-MHC complexes on the phone floor by means of T lymphocytes. Written within the hugely profitable Methods in Molecular Biology™ sequence structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, simply reproducible laboratory protocols, and key pointers on troubleshooting and averting identified pitfalls.

Authoritative and useful, Antigen Processing: equipment and Protocols is designed for rookies and specialists drawn to learning antigen processing.

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For an exact measurement of the activity, a kinetic analysis is preferred over endpoint assays (see Note 8). 1. 3, 1, 3, 10, 30 μM) in triplicates. 2. 5 μg of purified proteasomes in 200 μl of FSB containing either 200 μM of either suc-LLVY-AMC, or Z-ARRAMC, or Z-LLE-AMC. Prepare triplicates for each fluorogenic substrate. 3. Measure fluorescence intensity of each sample every 2 min in a fluorimeter (incubation temperature set to 37°C, excitation at 360 nm, emission at 450 nm) for a period of 100 min.

5. Due to the complexity of the samples it is thus possible to monitor the conditions of measurement via LC and MS. It is not intended to quantify fragments. 9GPS standard derived from pp89 is a peptide, which is not generated in the proteasomal digestion of pp89. This fragment is added prior the MS analysis and serves as a standard. During the whole analysis the peak area of 9GPS in comparable samples should be approximately the same. In addition, the amount of standard must be high enough to generate significant MSMS.

In addition, the amount of standard must be high enough to generate significant MSMS. 6. For 150–200 μg proteasome 109–1010cells should be used. 7. It is important to eliminate the ethanol. 8. A280 control: The absorbance at 280 nm is applicable to calculate the amount of protein. 2 Analysis of Proteasome Generated Antigenic Peptides by Mass Spectrometry 25 9. The 20S proteasome is precipitated between 38 and 80% saturation of (NH4)2SO4. 10. 5 ml 40% sucrose in the retral chamber (the higher density solution is underlayed to the lower density solution).

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