Frozen samples were immersed in a lysis buffer comprising 75 mM NaCl (Sigma Aldrich), 3% sodium dodecyl sulfate (Fisher Scientific), 1 mM sodium fluoride (VWR International, LLC), 1 mM β-glycerophosphate (Sigma Aldrich), 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate (VWR International, LLC), 1 mM phenylmethylsulfonyl fluoride (Fisher Scientific), 50 mM Hepes (Fisher Scientific) pH 8.5, and 1× complete ethylenediaminetetraacetic acid-free protease inhibitor mixture. Samples were subjected to rapid mixing and probe sonication using a Q500 QSonica sonicator (Qsonica) equipped with a 1.6-mm microtip at amplitude 20%. Samples were subjected to three cycles of 10 s of sonication followed by 10 s of rest, with a total sonication time of 50 s.
Sodium orthovanadate
Manufactured by Avantor
Sourced in Germany
Sodium orthovanadate is a chemical compound that serves as a common laboratory reagent. It is a white crystalline solid with the chemical formula Na3VO4. Sodium orthovanadate is used in various biochemical and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Q500 sonicator, by Qsonica (1 mentions)
Sodium dodecyl sulfate (sds), by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Phenylmethylsulfonyl fluoride, by Thermo Fisher Scientific (1 mentions)
Sodium fluoride, by Avantor (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Supersome, by Corning (1 mentions)
Methanol, by Avantor (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Trizma base, by Merck Group (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Atp magnesium salt, by Merck Group (1 mentions)
Indinavir, by Merck Group (1 mentions)
Nelfinavir, by Merck Group (1 mentions)
Sulfasalazine, by Merck Group (1 mentions)
Ritonavir, by Merck Group (1 mentions)
Sulfasalazine, by Avantor (1 mentions)
Amprenavir, by Merck Group (1 mentions)
Saquinavir mesylate, by Merck Group (1 mentions)
2 protocols using sodium orthovanadate
1
Lysis and Sonication for Protein Extraction
2
Baculovirus-Expressed BCRP Transporter Assay
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The baculovirus-infected insect cell microsomes (Supersomes) containing human complementary DNA-expressed BCRP (Arg482, 5 mg protein/mL) and wildtype Supersomes without hBCRP (control membrane, 5 mg protein/mL) used as negative control were obtained from Corning (Amsterdam, The Netherlands). After delivery, Supersomes were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at -80 °C until use.
AMP disodium salt, ADP sodium salt, ATP magnesium salt, guanosine 5'diphospate (GDP) sodium salt, uridine 5'-phosphate (UDP) sodium salt hydrate, sulfasalazine, sodium orthovanadate, amprenavir, indinavir, nelfinavir, ritonavir, saquinavir mesylate, ammonium acetate, MES hydrate, and Trizma base were obtained from Sigma-Aldrich (Taufkirchen, Germany), formic acid (MS grade) from Fluka (Neu-Ulm, Germany), acetonitrile, methanol (both LC-MS grade), and all other chemicals from VWR (Darmstadt, Germany).
Stock solutions were prepared in bidistilled water for sodium orthovanadate (10 mM), AMP, ADP, ATP, GDP, and UDP (20 mM, respectively) or in methanol for sulfasalazine (0.5 mg/mL), amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir (1 mg/mL, respectively). Stock solutions were aliquoted and stored at -20 °C until use.
AMP disodium salt, ADP sodium salt, ATP magnesium salt, guanosine 5'diphospate (GDP) sodium salt, uridine 5'-phosphate (UDP) sodium salt hydrate, sulfasalazine, sodium orthovanadate, amprenavir, indinavir, nelfinavir, ritonavir, saquinavir mesylate, ammonium acetate, MES hydrate, and Trizma base were obtained from Sigma-Aldrich (Taufkirchen, Germany), formic acid (MS grade) from Fluka (Neu-Ulm, Germany), acetonitrile, methanol (both LC-MS grade), and all other chemicals from VWR (Darmstadt, Germany).
Stock solutions were prepared in bidistilled water for sodium orthovanadate (10 mM), AMP, ADP, ATP, GDP, and UDP (20 mM, respectively) or in methanol for sulfasalazine (0.5 mg/mL), amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir (1 mg/mL, respectively). Stock solutions were aliquoted and stored at -20 °C until use.
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