Shotgun proteomics (mouse)
Pellets of membrane-protected material were lysed with RIPA buffer containing quenchers (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1x cOmplete Protease Inhibitor Cocktail (Roche), 1x PhosSTOP 956 (Roche), 10 mM sodium ascorbate, 1 mM Trolox and 1 mM sodium azide), sonicated and centrifuged at 10,000x g for 10 min. The supernatant was incubated with Streptavidin-agarose (Sigma-Aldrich) overnight, which was balanced with RIPA buffer containing quenchers. After 3x wash with RIPA buffer and 3x wash with 3 M Urea dissolved in 50 mM NH4HCO3, beads were incubated TCEP (5 mM, Sigma-Aldrich) at 55 °C for 30 min and shaken at 1000x rpm. Samples were alkylated with IAA (10 mM, Sigma-Aldrich) at room temperature for 20 min and shaken at 1000x rpm, further quenched by DTT (20 mM, Sigma-Aldrich) and washed 2x with 2 M Urea dissolved in 50 mM NH4HCO3. After overnight incubation with trypsin (1 μg/20 μl beads, Promega), supernatants were collected, plus 2x washes with 2 M Urea buffer. The samples were 966 acidified with trifluoroacetic acid (1%) and underwent vacuum centrifugation to decrease the volume. After being desalted on C18 stage tips (Thermo Scientific), peptides were reconstituted with 0.5% acetic acid for mass spectrometry analysis.
SEEK ID: http://lmmeisd-2.srv.mwn.de/assays/51
Experimental assay
Projects: Published Datasets
Investigation: Proteomics (Published)
Assay position:
Assay type: Proteomics
Technology type: Technology Type
Organisms: Mus musculus
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Creators and SubmitterViews: 56
Created: 9th Jul 2024 at 13:12
Last updated: 15th Oct 2024 at 12:07
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Projects: Published Datasets, Unpublished Datasets
Institutions: LMU Klinikum
Research Data Steward
This project serves as a centralized repository for omics datasets published by research groups within the SyNergy Cluster. It encompasses investigations such as proteomics and transcriptomics, which are further divided into individual studies led by SyNergy members. Each study is linked to relevant publications, assays and data files (with links to external repositories).
To explore investigations and their associated studies in more detail, please visit the 'Related items' tab on the Project ...
Public web page: Not specified
Submitter: Rainer Malik
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Assays: Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Bottom-up proteomics (human), Bottom-up proteomics (mouse), Bottom-up proteomics (mouse), Bottom-up proteomics (mouse), Bottom-up proteomics (mouse), Gel-based experiment (human), Phosphoproteomics / Bottom-up proteomics (mouse), Proximity-proteomics-based autophagosome content profiling (human), SWATH MS (human), SWATH MS (human, mouse), SWATH MS (mouse), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human, mouse), Shotgun proteomics (human, mouse), Shotgun proteomics (human, mouse), Shotgun proteomics (human, mouse), Shotgun proteomics (macaque), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (rat), Untargeted Proteomics (mouse)
CD4+ T cells orchestrate both humoral and cytotoxic immune responses. While it is known that CD4+ T cell proliferation relies on autophagy, direct identification of the autophagosomal cargo involved is still missing. Here, we created a transgenic mouse model, which, for the first time, enables us to directly map the proteinaceous content of autophagosomes in any primary cell by LC3 proximity labelling. IL-7Rα, a cytokine receptor mostly found in naïve and memory T cells, was reproducibly detected ...
CD4+ T cells orchestrate both humoral and cytotoxic immune responses. While it is known that CD4+ T cell proliferation relies on autophagy, direct identification of the autophagosomal cargo involved is still missing. Here, we created a transgenic mouse model, which, for the first time, enables us to directly map the proteinaceous content of autophagosomes in any primary cell by LC3 proximity labelling. IL-7Rα, a cytokine receptor mostly found in naïve and memory T cells, was reproducibly detected ...
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