In order to generate HLA-A*02:01 (MHC-I) monomers, the corresponding α-chain and β2m protein constructs were over-expressed separately in E.coli. The protein was refolded from the inclusion bodies in the presence of a UV-cleavable peptide and biotinylated for downstream applications as described previously(63 (link)). After purification, the protein was concentrated and stored with 20% glycerol at −80°C. For each epitope specificity tested in this study, peptide exchange reactions were set up in a volume of 100μl containing 0.2mM peptide and 100μg/ml HLA-A*02:01 protein in PBS (pH 7.4). The reaction mixture was exposed to 365nm UV-light irradiation for 20 mins using a Stratagene UV Stratalinker 2400 in 96-well U-shaped-bottom microplates (Corning). The plate was then transferred to 4°C overnight to complete the exchange. The protein was subsequently buffer exchanged against PBS (pH 7.4) using Microcon centrifugal filters (10kDa cut-off, MilliporeSigma) to remove the excess free peptide and subsequently spun at 13000×g for 15mins at 4°C to remove aggregates. The protein was filtered and stored at 4°C until further use.
Stratagene uv stratalinker 2400
Manufactured by Corning
The Stratagene UV Stratalinker 2400 is a compact and efficient UV crosslinking system designed for a range of laboratory applications. It features a microprocessor-controlled timer and adjustment knob for precise control over exposure time. The Stratalinker 2400 emits UVC radiation, which is commonly used for nucleic acid crosslinking and other experimental procedures requiring controlled UV exposure.
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Lab products found in correlation
2 protocols using stratagene uv stratalinker 2400
1
Generating HLA-A*02:01 Monomers from E. coli
2
Recombinant HLA-A*02:01 Monomer Production
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To generate HLA-A*02:01 (MHC-I) monomers, the corresponding α-chain and β2m protein constructs were overexpressed separately in E. coli. The protein was refolded from the inclusion bodies in the presence of an ultraviolet (UV)–cleavable peptide and biotinylated for downstream applications as described previously (63 (link)). After purification, the protein was concentrated and stored with 20% glycerol at −80°C. For each epitope specificity tested in this study, peptide exchange reactions were set up in a volume of 100 μl containing 0.2 mM peptide and HLA-A*02:01 protein (100 μg/ml) in PBS (pH 7.4). The reaction mixture was exposed to 365 nm UV light irradiation for 20 min using a Stratagene UV Stratalinker 2400 in 96-well U-shaped bottom microplates (Corning). The plate was then transferred to 4°C overnight to complete the exchange. The protein was subsequently buffer-exchanged against PBS (pH 7.4) using Microcon centrifugal filters (10-kDa cutoff, MilliporeSigma) to remove the excess free peptide and subsequently spun at 13,000g for 15 min at 4°C to remove aggregates. The protein was filtered and stored at 4°C until further use.
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