Amicon Ultra-4 centrifugal filters (3kDa cut-off) came from Merck Millipore. Constituents of the Protease inhibitor cocktail [41 (link),57 (link)] were purchased from Sigma-Aldrich (St. Louis) and AMRESCO Inc. (Dallas, TX, USA). All electrophoresis grade chemicals used in two-dimensional gel electrophoresis (2DE) were purchased from AMRESCO Inc. 3-10NL IPG strips, tributyl phosphine and Polyvinylidene difluoride (PVDF) membrane (0.2 µM pore size) were from Bio-Rad (Hercules, CA, USA). Anti-human rabbit polyclonal iPLA2 antibody (PA5-27945) and anti-human mouse monoclonal sPLA2 antibody (ab-24498) were from Invitrogen and Abcam (Cambridge, UK), respectively. Blocking agents―non-fat dry milk powder and BSA―were from Devondale (Saputo Dairy, Australia) and Sigma Aldrich, respectively. HRP linked anti-mouse IgG (NA93IV) and anti-rabbit IgG (NA934VS) antibodies came from GE Healthcare (Buckinghamshire, UK). Lumunata Cresendo Western HRP substrate was purchased from Merck Millipore. The blots and gels were imaged using the Image Quant™ LAS 4000 Biomolecular Imager (GE Healthcare, Chicago, Il, USA).
Non fat dry milk powder
Manufactured by Merck Group
Sourced in Australia, Germany
Non-fat dry milk powder is a dehydrated form of milk that has had the fat content removed. It serves as a powdered ingredient for various food and beverage applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti rabbit igg na934v, by GE Healthcare (1 mentions)
Amicon ultra 4 centrifugal filter, by Merck Group (1 mentions)
Tributyl phosphine, by Bio-Rad (1 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Image studio lite 5, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Ecl plus western blot detection system, by GE Healthcare (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
Cortisone, by Merck Group (1 mentions)
Monopotassium phosphate, by Merck Group (1 mentions)
Cortisol, by Merck Group (1 mentions)
Monosodium phosphate, by Merck Group (1 mentions)
Lichrospher 100 rp 18, by Merck Group (1 mentions)
4 protocols using non fat dry milk powder
1
Proteomic analysis with PLA2 antibodies
2
Quantitative Western Blot Analysis of EHMT1
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Extracts of cells terminally differentiated for 10 days were obtained by applying RIPA lysis buffer (50 mm Tris, pH 8.0; 150 mm NaCl, 1% NP-40, 0.1% SDS, 10% glycerol, 9.8 mm NaF and 1% protease inhibitor cocktail; Merck-Millipore). Samples containing equal amounts of proteins (30 μg per lane) were subjected to SDS-PAGE (NuPAGE Novex Bis-Tris Gel; 4–12% Thermo Fisher Scientific) and blotted onto PVDF membrane (Merck-Millipore). After blocking with 5% w/v non-fat dry milk powder in 0.1% Tween 20 (Sigma-Aldrich), membranes were probed with antibodies specific for the N-terminal of the EHMT1 protein (ab135487, Abcam) and for GAPDH as control (Sigma-Aldrich). Signals were visualized with horseradish peroxidase-coupled anti-rabbit IgG antibody (Santa Cruz Biotechnology, Dallas, TX, USA) using ECL Plus Western Blot Detection System (GE Healthcare Europe, Freiburg, Germany). Intensity values were calculated using Image Studio Lite 5.2 software (LI-COR Biotechnology, Bad Homburg, Germany). Western blot analysis was performed on at least three independent biological replicates.
3
Quantifying Cortisol and Catechin Compounds
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Cortisol, cortisone and EGCG were purchased from Sigma-Aldrich (St. Louis, MO). The following chemicals were procured from Carl Roth GmbH + Co. KG (Karlsruhe, Germany): Ethylacetate, NADP, NADPH, non-fat dry milkpowder, monosodium phosphate and potassium chloride, HPLC column LiChrospher® 100 RP-18, methanol, sodium chloride and monopotassium phosphate were obtained from Merck (Darmstadt, Germany). (−)-Epicatechin gallate, (−)-catechin and (−)-gallocatechin were purchased from Biomol GmbH (Hamburg, Germany), Cayman Chemical (Ann Arbor, MI) and LKT Laboratories Inc. (St. Paul, MN), respectively. The different types of commercially available teas consisting of green, oolong and black teas were bought from supermarkets in Kiel, Germany. The anti-HSD11B1 antibody (ab83522) was obtained from Abcam (Cambridge, UK).
4
Quantitative Western Blot Analysis
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Total protein was extracted from the cells using RIPA lysis buffer (Beyotime Ltd., Beijing, China), quantified using a BCA protein assay kit (Bio-Rad Laboratories, Hercules, USA) and separated via a 12% SDS-PAGE (NuPAGE; Invitrogen; Thermo Fisher Scientific, Inc.). The separated proteins were subsequently transferred onto a PVDF membrane (Whatman plc; GE Healthcare Life Sciences) and blocked with 5% non-fat dry milk powder (Merck KGaA) at room temperature for 2 h. The PVDF membrane was then incubated with RCBTB1 (1:1000, Immunoway, YT0974), SLC1A5 (1:1000, Immunoway, YT7482), WNT7B (1:1000, Immunoway, YN0288) and Tubulin (1:5000, Abcam, ab7291) primary antibodies at 4°C overnight. Following the primary antibody incubation, the membranes were washed with TBS-Tween 20 three times and incubated with a goat anti-rabbit secondary antibody (1:3000, Bioss, Beijing China) for 2 h at room temperature. Protein bands were visualized using a chemiluminescence reagent on a gel imaging system. The gray value of the target protein was normalized to the gray value of the internal reference protein. Tubulin and the data are presented as the relative content of the target protein in a sample.
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