TC32 cells (1.5 × 106) were incubated with drug, collected, washed with PBS, lysed, and boiled in 4% LDS buffer (0.125 M Trizma hydrochloride buffer solution, pH 7.5, and 4% lithium dodecyl sulfate (Sigma-Aldrich). Protein concentrations were determined after diluting the detergent using the bicinchoninic acid (BCA) assay kit (Pierce Protein Biology Products, Carlsbad, CA). Thirty micrograms of protein was resolved on a 4–12% NuPAGE Bis-Tris Mini gels (Invitrogen, Carlsbad, CA) in 1 × 4-morpholinepropanesulfonic acid (MOPS) sodium dodecyl sulfate (SDS) buffer (Invitrogen), and transferred to nitrocellulose (GE Healthcare Life Sciences, Pittsburgh, PA). The membrane was probed with the following antibodies: mouse monoclonal anti-FLI1 (1:1,000, Abcam, Cambridge, MA), mouse polyclonal anti-ACTB (1:1,000, Cell Signaling, Danvers, MA), rabbit polyclonal anti-ENT1 (1:500, Abcam), rabbit polyclonal anti-ENT2 (1:1000, Abcam), and rabbit polyclonal anti-TK1 (1:000, Santa Cruz Biotechnology, Dallas, TX). The protein was visualized by using horseradish peroxidase (HRP)-conjugated secondary antibody and ECL (Amersham, Buckinghamshire, UK).
Lithium dodecyl sulfate (lds)
Manufactured by Merck Group
Sourced in United Kingdom
Lithium dodecyl sulfate is a chemical compound used as a surfactant and detergent in various laboratory applications. It functions as a wetting agent, emulsifier, and solubilizer, helping to disperse and suspend materials in liquid solutions.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris mini gel, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose, by GE Healthcare (2 mentions)
Mouse monoclonal anti fli1, by Abcam (2 mentions)
Mouse polyclonal anti actb, by Cell Signaling Technology (2 mentions)
Mutanolysin, by Merck Group (2 mentions)
Lysozyme, by MP Biomedicals (2 mentions)
Lysostaphin, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Teknova (2 mentions)
Ethylene diamine tetraacetic acid (edta), by New England Biolabs (2 mentions)
Proteinase k, by New England Biolabs (2 mentions)
Zymolyase, by Zymo Research (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Pepsin, by Merck Group (1 mentions)
Trizma hydrochloride buffer solution, by Merck Group (1 mentions)
Hela cells, by European Collection of Cell Cultures (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Reducing agent, by Merck Group (1 mentions)
Fuji film intelligent dark box 2, by Fujifilm (1 mentions)
β mercaptoethanol, by Bio-Rad (1 mentions)
7 protocols using lithium dodecyl sulfate (lds)
1
Western Blot Analysis of Cell Signaling
2
Multiplexed PLISH Imaging Protocol
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Multiplex fluorescence in situ hybridisation was performed using PLISH (Nagendran et al. 2018 (link)). Sections were de-paraffinised, boiled in a 10 mM citrate buffer (pH 6.0) with 0.05% lithium dodecyl sulfate (Sigma) and processed in sealed hybridisation chambers (Grace Biolabs, Bend, US). Tissues were treated with 0.1 mg/mL Pepsin (Roche-10108057001, Sigma-Aldrich) in 0.1 M HCl, followed by (at 37°C): hybridisation of barcoded gene probes and bridge sequences, DNA ligation (10 CEU/mL T4 DNA ligase, M0202T, New England Biolabs, Ipswich, US) and extension by rolling circle amplification (1 U/mL Nxgen phi29 polymerase, Lucigen, Middleton, US). Samples were incubated with fluorophore-conjugated oligonucleotides specific to the barcode for the targeted gene probe (Supplementary Methods, see section on supplementary materials given at the end of this article), washed then treated with TruVIEW autofluoresence quenching kit (Vector Laboratories, Burlingame, US), stained with DAPI and mounted. Imaging was performed with a Zeiss Axio Imager M1 microscope (Jena, Germany) and analysed with Qupath software (Bankhead et al. 2017 (link)).
3
Western Blot Analysis of Protein Expression
4
Microgel Cell Lysis and Protein Removal
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To lyse the cells in the microgels, the particles are submerged in a solution of 2 mL TE buffer solution containing 10 mM DTT (manu), 2.5 mM EDTA (Teknova), and 10mM NaCl (Sigma-Aldrich). The following quantities of lytic enzymes are also included: 4 U zymolyase (Zymo Research), 10 U lysostaphin (Sigma-Aldrich, catalog no. L7386), 100 U mutanolysin (Sigma-Aldrich, catalog no. M9901), and 40 mg lysozyme (MP Biomedicals, catalog no. 195303). Cell lysis proceeds overnight in a shaking incubator at 37°C. The turbid lysate mixture is centrifuged at 1000 g for 1 min, the supernatant removed, and 3 mL of a solution containing 0.5% (w/v) lithium dodecyl sulfate (Sigma-Aldrich) and 10 mM EDTA in TE buffer is added, along with 4 U of Proteinase K (NEB) to solubilize cell debris and digest cellular proteins. The solution is incubated at 50°C on a heating block for 30 min. Following lysis, the microgels are thoroughly washed to ensure complete removal of detergents and other chemical species which may inhibit downstream molecular biology reactions. The following washes occur in 10 mL volumes with centrifugation magnitudes of 1000 g between additions of wash solutions: one wash with 2% (v/v) Tween 20 in water; one wash in 100% ethanol (Koptec) to denature any remaining Proteinase K; and five washes with 0.02% (v/v) Tween 20 in water.
5
Microgel Cell Lysis and Protein Removal
6
Culturing HeLa Cells in Media
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Human epithelial carcinoma (HeLa) cells were obtained from the European Collection of Cell Cultures (Salisbury, UK) and maintained in DMEM supplemented with GlutaMAX I and 10% fetal bovine serum (FBS, Invitrogen), 10% charcoal stripped FBS (CSS, Gibco), or lipoprotein deficient FBS (LDS, Sigma) in a humidified atmosphere of 5% CO2 at 37 °C.
7
SDS-PAGE Protein Separation Protocol
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SDS-PAGE was performed using precast Bis-Tris 12% polyacrylamide gels and an electrophoresis system (Invitrogen, Mount Waverley, Australia). Centrifugal supernatants were diluted (1:25) with deionized water and mixed (10 µl) with 5 µl of LDS (Sigma Aldrich, UK), 2 µl of reducing agent (Sigma Aldrich, UK) and 5 µl of β-mercaptoethanol (Bio-Rad Laborato-ries Ltd., UK). Each of the non-heated reference samples was diluted in a similar way and the same amount of reagents was added. All samples were heated at 100 °C for 3 min prior to loading. The gels were run at 115 V for 90 min and a solution containing 0.025% w/v Coomassie (Bio-Rad Laboratories Ltd., Watford, UK), 40% w/w methanol and 7.5% w/w acetic acid was used for staining for 90 min and a 7.5% w/w acetic acid solution was used for destaining the gels overnight. Gels were visualized using a Fuji Film Intelligent Dark Box II with Fuji Film LAS-3000 V2.2 software (Brookvale, Australia). The proteins were identified by comparison to the molecular weight (Mw 10-250 kDa) of protein standards (Precision Plus, Kaleidoscope, BioRad, Australia).
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