1 μL of drug in 100% DMSO was dispensed to each well of a 96-well round-bottom culture plate utilizing the Labcyte ECHO acoustic liquid handler. 2 × 107 MACS-purified parasites in 199 μL of RPMIc were then added to each well. Parasites were incubated with drug for 2 h at 37 °C, 5% CO2, 5% O2. Next, samples were transferred from 96-well plates to 1.5 mL screw-cap microfuge tubes. 35 μCi of EasyTag™ Express S35 Protein Labeling Mix (Perkin Elmer) diluted to 10 μL with RPMIc was added to each tube. Reactions were incubated at 37 °C with mild shaking for 2 h.
Easytag express s35 protein labeling mix
Manufactured by PerkinElmer
The EasyTag™ Express S35 Protein Labeling Mix is a laboratory reagent used for the metabolic labeling of newly synthesized proteins. It contains the radioactive isotope sulfur-35 (S35) which can be incorporated into proteins during translation.
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Lab products found in correlation
2 protocols using easytag express s35 protein labeling mix
1
Metabolic Labeling of Parasite Cultures
2
Pulse-Chase Protein Labeling Assay
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Cells were incubated with cysteine/methionine-free DMEM supplemented with 165 mCi of EasyTag EXPRESS S35 protein labeling mix (PerkinElmer) for 30 min. Cells were then lysed with RIPA buffer followed by centrifugation. Lysates were spotted on Whatman paper. Protein was precipitated with cold 5% TCA and washed with 10% TCA, ethanol, and acetone. The Whatman paper was air dried for 10 min and dipped into 8 mL scintillation cocktail. Radioactivity was measured using LS6500 Scintillation counter and normalized with cell number.
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