All cell culture reagents were purchased from Gibco-Life Technology (Eggenstein, Germany). Sch A was from MedChem Express (Monmouth Junction, NJ, USA). DON was from Sigma Chemical Company (St. Louis, MO, USA). They were dissolved in dimethylsulfoxide (DMSO) (Sigma) and stored at −20 °C before use. Phosphate buffered saline (PBS), sodium biocarbonate, diethylpyrocarbonate (DEPC) and chloroform were purchased from Sigma. Ammonium persulfate, N, N, N′, N′-tetramethyl-ethane-1,2-diamine (TEMED), acrylamide, resolving gel buffer, stacking gel buffer, 10% (w/v) Tween 20 and 20% (v/v) sodium dodecyl sulfate (SDS) were purchased from Biorad (Richmond, CA, USA). 30% (w/w) hydrogen peroxide (H2O2) solution, absolute ethanol, and isopropanol were from Merck (Darmstadt, Germany). RNAisoPlus was purchased from Takara (Otsu, Japan). HiScriptTM RT SuperMix for qPCR and AceQ qPCR SYBR Green Master Mix were obtained from Vazyme Biotech Co. (Piscataway, NJ, USA).
Stacking gel buffer
Manufactured by Bio-Rad
Sourced in United States
Stacking gel buffer is a laboratory solution used in electrophoresis techniques, specifically in the preparation of polyacrylamide gels. Its core function is to create a pH gradient that facilitates the stacking of proteins or other macromolecules at the interface between the stacking and resolving gels, prior to separation.
Automatically generated - may contain errors
Lab products found in correlation
Resolving gel buffer, by Bio-Rad (2 mentions)
Absolute ethanol, by Merck Group (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Sodium dodecyl sulfate (sds), by Bio-Rad (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Ammonium persulfate, by Bio-Rad (1 mentions)
Hiscript rt supermix for qpcr, by Vazyme (1 mentions)
Sch a, by MedChemExpress (1 mentions)
Diethylpyrocarbonate depc, by Merck Group (1 mentions)
Protein marker, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Protein sample buffer, by Bio-Rad (1 mentions)
Acrylamide bis acrylamide solution, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
3 protocols using stacking gel buffer
1
Cell Culture Reagents and Protocols
2
Hormone Assay and Western Blot Protocol
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E2 and P4 hormone radioactive assay kits were purchased from ICN (ICN Biomedicals, Inc., Costa Mesa, CA, USA), the PVDF membrane and ECL were from Luminex xMAP - EMD Millipore (MA, USA), goat anti-rabbit IgG (H + L), goat anti-mouse IgG (H + L), HRP and rabbit anti-goat IgG (H + L) were from Jackson Immunoresearch Laboratories (PA, USA); protein extraction RIPA was from Thermo Fisher Scientific (MA, USA); the MT1 antibody (SC-390328; Santa Cruz Bio Inc., Santa Cruz, CA, USA), MT2 antibody (SC-13177; Santa Cruz Bio Inc.) and p53 antibody (SC-47698; Santa Cruz Bio Inc.) were from Santa Cruz Biotechnology; the protein marker was from Thermo Fisher Scientific; and the separation gel buffer, stacking gel buffer, 20% Tween 20 and protein sample buffer were from Bio-Rad Lab (CA, USA). Melatonin and other reagents, unless specified, were purchased from Sigma Chemical Co. (MO, USA).
3
Western Blot Analysis of TET1 and TET3
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Protein lysates were prepared using AllPrep DNA/RNA/Protein Kit (Qiagen), according to the manufacturer’s instructions. Polyacrylamide gels were poured in 8 % concentration using 30 % acrylamide/bisacrylamide solution (Bio-Rad), sodium dodecyl sulfate (SDS) (Bio-Rad), ammonium persulfate (APS) (Bio-Rad), tetramethylethylenediamine (TEMED) (Bio-Rad), Resolving Gel Buffer (Bio-Rad) and Stacking Gel Buffer (Bio-Rad). Electrophoresis of protein lysates was performed using 10 µL of each sample, followed by Ponceau S staining. Samples were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The following antibodies were used for the detection of TET1, TET3, and beta-actin: anti-TET1 in dilution of 1:1000 (Thermo Fisher, PA5-72805), anti-TET3 in dilution 1:1000 (Abcam, ab 139311) and anti-beta-actin in dilution of 1:10 000 (Cell Signaling, 4967L). All membranes were incubated with a secondary anti-rabbit IgG antibody in a dilution of 1:7500, conjugated to horseradish peroxidise (HRP) (Thermo Fisher). Protein signals were detected by chemiluminescence using Clarity Max Western ECL Substrate (Bio-Rad) and quantified with ImageLab software (Bio-Rad). TET1 and beta-actin measurements were normalized to whole-cell lysate content based on the Ponceau S staining.
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