Lysis:Article Title: Target identification using drug affinity responsive target stability (DARTS).
Article Snippet: .. M-PER lysis buffer (Pierce, cat. no. 78501) Phosphatase inhibitor solutions (100 mM β-pyrophosphate, 50 mM sodium pyrophosphate, 200 mM sodium vanadate, and 1 M sodium fluoride) 20× Protease inhibitor solution (see recipe) Phosphate-buffered saline [PBS; 1× PBS was diluted from 10× PBS (MPbio, cat. no. PBS10X02) using filter-sterilized distilled water] 10-cm dish cell culture in logarithmic growth phase (up to 70% to 80% confluent) 10× TNC buffer (see recipe) BCA protein concentration assay (Pierce, cat. no. 23225 or similar assay) Dimethyl sulfoxide (DMSO; or other corresponding vehicle) 100× stock solution of small molecule(s) in DMSO (DMSO can be replaced with other vehicle of choice) Pronase stock solution (see recipe) 1× TNC buffer (dilute 10× TNC buffer 10-fold in dH2O) Cell scraper (e.g., Fisher, cat. no. 08-100-241) 1.5-ml tubes Refrigerated centrifuge capable of 18,000 × g (e.g., Beckman Microcentrifuge 22R) ..
Protease Inhibitor:Article Title: Target identification using drug affinity responsive target stability (DARTS).
Article Snippet: .. M-PER lysis buffer (Pierce, cat. no. 78501) Phosphatase inhibitor solutions (100 mM β-pyrophosphate, 50 mM sodium pyrophosphate, 200 mM sodium vanadate, and 1 M sodium fluoride) 20× Protease inhibitor solution (see recipe) Phosphate-buffered saline [PBS; 1× PBS was diluted from 10× PBS (MPbio, cat. no. PBS10X02) using filter-sterilized distilled water] 10-cm dish cell culture in logarithmic growth phase (up to 70% to 80% confluent) 10× TNC buffer (see recipe) BCA protein concentration assay (Pierce, cat. no. 23225 or similar assay) Dimethyl sulfoxide (DMSO; or other corresponding vehicle) 100× stock solution of small molecule(s) in DMSO (DMSO can be replaced with other vehicle of choice) Pronase stock solution (see recipe) 1× TNC buffer (dilute 10× TNC buffer 10-fold in dH2O) Cell scraper (e.g., Fisher, cat. no. 08-100-241) 1.5-ml tubes Refrigerated centrifuge capable of 18,000 × g (e.g., Beckman Microcentrifuge 22R) ..
Saline:Article Title: Target identification using drug affinity responsive target stability (DARTS).
Article Snippet: .. M-PER lysis buffer (Pierce, cat. no. 78501) Phosphatase inhibitor solutions (100 mM β-pyrophosphate, 50 mM sodium pyrophosphate, 200 mM sodium vanadate, and 1 M sodium fluoride) 20× Protease inhibitor solution (see recipe) Phosphate-buffered saline [PBS; 1× PBS was diluted from 10× PBS (MPbio, cat. no. PBS10X02) using filter-sterilized distilled water] 10-cm dish cell culture in logarithmic growth phase (up to 70% to 80% confluent) 10× TNC buffer (see recipe) BCA protein concentration assay (Pierce, cat. no. 23225 or similar assay) Dimethyl sulfoxide (DMSO; or other corresponding vehicle) 100× stock solution of small molecule(s) in DMSO (DMSO can be replaced with other vehicle of choice) Pronase stock solution (see recipe) 1× TNC buffer (dilute 10× TNC buffer 10-fold in dH2O) Cell scraper (e.g., Fisher, cat. no. 08-100-241) 1.5-ml tubes Refrigerated centrifuge capable of 18,000 × g (e.g., Beckman Microcentrifuge 22R) ..
Cell Culture:Article Title: Target identification using drug affinity responsive target stability (DARTS).
Article Snippet: .. M-PER lysis buffer (Pierce, cat. no. 78501) Phosphatase inhibitor solutions (100 mM β-pyrophosphate, 50 mM sodium pyrophosphate, 200 mM sodium vanadate, and 1 M sodium fluoride) 20× Protease inhibitor solution (see recipe) Phosphate-buffered saline [PBS; 1× PBS was diluted from 10× PBS (MPbio, cat. no. PBS10X02) using filter-sterilized distilled water] 10-cm dish cell culture in logarithmic growth phase (up to 70% to 80% confluent) 10× TNC buffer (see recipe) BCA protein concentration assay (Pierce, cat. no. 23225 or similar assay) Dimethyl sulfoxide (DMSO; or other corresponding vehicle) 100× stock solution of small molecule(s) in DMSO (DMSO can be replaced with other vehicle of choice) Pronase stock solution (see recipe) 1× TNC buffer (dilute 10× TNC buffer 10-fold in dH2O) Cell scraper (e.g., Fisher, cat. no. 08-100-241) 1.5-ml tubes Refrigerated centrifuge capable of 18,000 × g (e.g., Beckman Microcentrifuge 22R) ..
Protein Concentration:Article Title: Target identification using drug affinity responsive target stability (DARTS).
Article Snippet: .. M-PER lysis buffer (Pierce, cat. no. 78501) Phosphatase inhibitor solutions (100 mM β-pyrophosphate, 50 mM sodium pyrophosphate, 200 mM sodium vanadate, and 1 M sodium fluoride) 20× Protease inhibitor solution (see recipe) Phosphate-buffered saline [PBS; 1× PBS was diluted from 10× PBS (MPbio, cat. no. PBS10X02) using filter-sterilized distilled water] 10-cm dish cell culture in logarithmic growth phase (up to 70% to 80% confluent) 10× TNC buffer (see recipe) BCA protein concentration assay (Pierce, cat. no. 23225 or similar assay) Dimethyl sulfoxide (DMSO; or other corresponding vehicle) 100× stock solution of small molecule(s) in DMSO (DMSO can be replaced with other vehicle of choice) Pronase stock solution (see recipe) 1× TNC buffer (dilute 10× TNC buffer 10-fold in dH2O) Cell scraper (e.g., Fisher, cat. no. 08-100-241) 1.5-ml tubes Refrigerated centrifuge capable of 18,000 × g (e.g., Beckman Microcentrifuge 22R) ..
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