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 procedure known as T lymphocytes. In Antigen Processing: tools and Protocols, specialist researchers within the box offer a accomplished set of protocols for learning presentation of antigens produced within the ordinary processing pathways for MHC category I and sophistication II molecules. The chapters persist with chronology of intracellular processing occasions, finishing with attractiveness of peptide-MHC complexes on the phone floor by means of T lymphocytes. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and key pointers on troubleshooting and warding off identified pitfalls.
Authoritative and functional, Antigen Processing: tools and Protocols is designed for novices and specialists attracted to learning antigen processing.
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Additional info for Antigen Processing: Methods and Protocols
D) Fractions are collected in a volume of 1 ml during the elution with 20–40% buffer B (see Note 11). 1, step 6). 6. To prepare the proteasome activity assay: (a) The substrate (Suc-LLVY-AMC; 20 mM stock solution) measuring the chymotrypsin-like activity is added freshly to the proteasome assay buffer (10 μl to 10 ml buffer to a final concentration 20 μM). (b) 10 μl sample or 10 μl water (reference value) are placed into the wells of a black 96-well microtiter plate. 100 μl substrate assay buffer is added.
3. Due to high viscosity of the erythrocyte lysate, loading by gravity flow is not possible. When applying vacuum, be careful not to let the DEAE material run dry. The capacity of the DEAE-Cellulose is high enough to bind most of the 20S and 26S proteasomes. If the flow through is passed again over the batch adsorption column after regeneration of the material, 20–30% higher yield can be achieved. 4. Standard measuring wavelengths for AMC are excitation at 360 nm and emission at 450 nm, alternative wavelengths are 380 nm for excitation and 430 nm for emission.
The biological matrix affects the MS signal. The 0-h-value has to include buffer and enzyme. To stop the reaction as quickly as possible, TFA should be placed in the tube for the zero hour time point. Be careful with the TFA concentration, because a high concentration of salt is able to quench the MS signal. 15. The separation of individual substances is based on the differential distribution of samples in two phases. One phase (liquid, mobile) moves to another phase (liquid, stationary) and takes over the constituents with different speeds.