WebDec 18, 2024 · Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. For label-free experiments, Jurkat E6-1 cells (ATCC) were grown in Roswell Park Memorial Institute 1640 (RPMI) media (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin, and streptomycin (Gibco). For SILAC experiments human embryonic kidney 293T (HEK293) cells transfected with … See more Three biological replicates of SILAC-labeled HEK293T cells were labeled with biotin-phenol (BP) (Iris Biotech GmbH) at >80% confluency using previously described methods4. … See more Jurkat cells were lysed with 8 M urea, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA (Sigma), 2 μg/μl Aprotinin (Sigma-Aldrich), 10 μg/μl Leupeptin (Roche), and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma) at 4 … See more A polyclonal anti-biotin (ImmuneChem Pharmaceuticals Inc., cat no. ICP0615) was used for biotin peptide enrichment. Peptide spike-in samples were composed of varying amounts of biotinylated peptides spiked in to … See more Jurkat peptides were labeled with EZ link NHS-Biotin (Thermo Fisher Scientific). The biotin reagent was brought to RT. For labeling, 1 mg of peptide from Jurkat cell lysate was … See more
Production and evaluation of parathyroid hormone receptor1
WebNov 16, 2024 · Briefly, the cell medium was replaced by complete DMEM containing 500 μΜ final biotin-phenol (Iris biotech) or DMSO (control) and incubated for 30 min at 37 °C in a 5% CO 2 incubator. The APEX enzyme was induced by addition of 1 mM final H 2 O 2 diluted in 1XDPBS, in the medium, for 1 min at room temperature (RT). Following the … WebJul 15, 2024 · 1 INTRODUCTION. Heme peroxidases are very useful enzymes for biological applications owing to their great activity and broad substrate specificity. 1 For instance, one of the most widely utilized heme peroxidases, horseradish peroxidase (HRP), is routinely applied as the reporters of chemiluminescence-based western blot, 2 … daft punk helmet cross section
An Optimized Protocol for Proximity Biotinylation in
WebIris Biotech GmbH Biotin-AEEA-phenol Revision date: 12.01.2024 Product code: LS-3490 Page 6 of 7 No data available Specific effects in experiment on an animal No data available Practical experience 11.2. Information on other hazards Further information RTECS: No data available Caution! To the best of our knowledge the toxicological properties ... WebSep 18, 2024 · Download : Download full-size image Figure 2. APEX2-Mediated Proximity Biotinylation in Confluent and Filter-Grown MDCK-II Cells (A) Titration of biotin phenol … biochar used as insulation