Individual prostate lobes were dissected in PBS, washed, and homogenized in PBS using a pestle. An equal volume of Lysis Buffer 2 (R&D 895347) was added and the homogenate incubated at room temperature for 30 min with agitation. The lysate was centrifuged at 10,000g for 30 min at 4°C, and the supernatant removed. Protein levels in each extract was determined using the Quick Start Bradford 1x Dye Reagent (Bio-Rad 500–0205), equal amounts of total protein were loaded into each well of the Mouse/Rat HGF Quantikine ELISA Kit (R&D Systems, MHG00) and the plate processed according to manufacturer’s instructions. Each sample was analyzed in duplicate, and Hgf levels for each sample was normalized to total protein in each well.
Lysis buffer 2
Manufactured by R&D Systems
Sourced in United States
Lysis Buffer 2 is a reagent used for cell lysis, a process of breaking down the cell membrane and releasing the cellular contents. This buffer is designed to facilitate the extraction and purification of proteins, nucleic acids, and other biomolecules from cells.
Automatically generated - may contain errors
Lab products found in correlation
Quick start bradford 1x dye reagent, by Bio-Rad (2 mentions)
Mouse rat hgf quantikine elisa kit, by R&D Systems (2 mentions)
Tnf α quantikine hs elisa kit, by R&D Systems (1 mentions)
Il 1β quantikine elisa kit, by R&D Systems (1 mentions)
Il 6 quantikine elisa kit, by R&D Systems (1 mentions)
Multiplex assay, by Merck Group (1 mentions)
Colorimetric assay kit, by Cayman Chemical (1 mentions)
Adiponectin, by Merck Group (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Chod pap, by Roche (1 mentions)
4 protocols using lysis buffer 2
1
Quantification of Hepatocyte Growth Factor
2
Quantification of Hepatocyte Growth Factor
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Individual prostate lobes were dissected in PBS, washed, and homogenized in PBS using a pestle. An equal volume of Lysis Buffer 2 (R&D 895347) was added and the homogenate incubated at room temperature for 30 min with agitation. The lysate was centrifuged at 10,000g for 30 min at 4°C, and the supernatant removed. Protein levels in each extract was determined using the Quick Start Bradford 1x Dye Reagent (Bio-Rad 500–0205), equal amounts of total protein were loaded into each well of the Mouse/Rat HGF Quantikine ELISA Kit (R&D Systems, MHG00) and the plate processed according to manufacturer’s instructions. Each sample was analyzed in duplicate, and Hgf levels for each sample was normalized to total protein in each well.
3
Quantifying Cytokine Levels in Skin Tissue
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Cytokines were quantified using the following ELISA kits: the IL-1β Quantikine ELISA kit (R&D Systems, Minneapolis, MN), IL-6 Quantikine ELISA kit (R&D Systems, Minneapolis, MN), and TNF-α Quantikine HS ELISA Kit (R&D Systems, Minneapolis, MN). The skin tissue was excised and homogenized in PBS. The number of viable bacterial cells in the tissue was determined by counting the number of colonies formed on mannitol salt medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10 µg/mL ampicillin. Excised tissues were homogenized in lysis buffer 2 (R&D Systems, Minneapolis, MN). After preparation according to the manufacturers’ instructions, lysates were stored at − 80 °C until the quantification of IL-1β. Blood samples were obtained via cardiac puncture under inhalation anaesthesia. After incubation for two hours at room temperature, serum was prepared and stored at − 80 °C until the quantification of IL-6 and TNF-α.
4
Biomarkers of Metabolic Health
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Plasma levels of total cholesterol, triglycerides (Chod-Pap; Roche Diagnostic GmbH, Mannheim, Germany), nonesterified fatty acids (Wako Chemical, Neuss, Germany), and liver transaminases (Biotecnica, SP, Brazil) were assayed using enzymatic-colorimetric methods according to the manufacturers' instructions. Leptin, adiponectin (Merck Millipore, Darmstadt, Germany), C-reactive protein (IBL-America, Minneapolis, USA), TNFα, and IL-1β (R&D Systems, Minneapolis, USA) plasma concentrations were determined using ELISA. For analysis of IL-1β expression in liver tissue, 50-mg tissue samples were homogenized in 2 mL Lysis Buffer 2 (R&D Systems, Minneapolis, USA). IL-6 plasma levels were analyzed using a Multiplex Assay (Merck Millipore, Darmstadt, Germany). Protein carbonyl content in the liver was measured using a colorimetric assay kit (Cayman Chemical Company, Michigan, USA).
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