To quantify protein expression, cisplatin sensitive and resistant cells were plated in triplicates in 6-well (100,000 cells/well) or 12-well plates (50,000 cells/well) and allowed to expand for 72 h before trypsinization, PBS washing, and addition of Laemmli sample buffer [0.5 M Tris HCl at pH 6.8 (50-213-711; Fisher Scientific), 10% sodium dodecyl sulfate (SDS), 100 mM PMSF (329-98-6; Sigma-Aldrich) in isopropanol (190764-4x4l; Sigma), glycerol (G5516-1L; Sigma-Aldrich), and protease inhibitor (11836170001; Sigma-Aldrich) dissolved in ddH2O]. Lysates of cells were sonicated with a Sonic Dismembrator Model F60 (remote, 10 s at 50% power, then 2 min on ice, repeated three times; Fisher Scientific) and passed through a Hamilton syringe (14-824-663; Thermo Fisher Scientific) to dissociate proteins attached to chromatin. Subsequently, samples were centrifuged at 10,000 rpm for 10 min, and the supernatant was transferred to a new tube. The membranes were stained with one antibody at a time to ensure specificity. Quantification of proteins was carryout out with a BCA protein assay (23227; Thermo Fisher Scientific). Absorbance was measured at 562 nm using a SpectraMax i3x microplate reader (Molecular Devices LLC). Values were normalized to both the internal control (GAPDH or alpha-beta tubulin) and the average value of the control sample (PDX4 SE).
Sonic dismembrator model f60
Manufactured by Thermo Fisher Scientific
The Sonic Dismembrator Model F60 is a laboratory instrument designed for the disruption and homogenization of biological samples. It utilizes high-frequency sound waves to break down and homogenize samples, enabling efficient extraction and preparation of cellular components, proteins, nucleic acids, and other biomolecules for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Hamilton syringe, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Spectramax i3x microplate reader, by Molecular Devices (1 mentions)
2 protocols using sonic dismembrator model f60
1
Quantifying Protein Expression in Cisplatin-Sensitive and Resistant Cells
2
Intestinal Phosphate Measurement Protocol
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Mice on a control diet were euthanized 2 weeks after tamoxifen administration via isoflurane overdose. In another cohort, mice were administered tamoxifen, fed a control diet for 2 weeks, followed by an additional 2 weeks of high Pi diet and were then euthanized. After euthanasia, the small intestine and colon were removed. The small intestine was cut into 2 segments of equal length, labelled with proximal (closest to the stomach) and distal, respectively. Subsequently, the contents of the proximal and distal segments as well as of the colon were extruded into 5 mL tubes. The weight of the content was determined gravimetrically and 0.75 mol L−1 HNO3 was added equal to 5‐fold the weight of the contents. Then, the samples were homogenized by ultrasonication (Sonic Dismembrator Model F60, Fisher Scientific). After overnight incubation at room temperature, samples were centrifuged at 1,000g for 5 minutes to pellet debris. Then the supernatants were transferred into 1.5 mL tubes for centrifugation at 17,000g for 20 minutes. After centrifugation, the final supernatants were collected and analysed for Na+ and Pi, as described below.
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