SDS-PAGE was performed according to Laemmli's method (Laemmli, 1970 (link)). Briefly, 20 μg of the protein sample mixed with nonreducing 5× Protein Loading Buffer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was incubated for 5 min at 100°C and then loaded into a 12% SurePAGE precast gel (GenScript, New Jersey, United States). Protein samples were separated in Tris-MOPS-SDS running buffer (GenScript, New Jersey, United States) in a Bio-Rad Mini-PROTEAN Tetra System (Bio-Rad, California, United States) at 120 V for approximately 90 min. Gels were stained with 0.5% Coomassie brilliant blue R-250 and then photographed and analyzed by a Bio-Rad Gel Doc EZ Imager (Bio-Rad, California, United States). The low molecular standard (Yuanye, Shanghai, China) includes rabbit phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; rabbit actin, 43.0 kDa; bovine carbonic anhydrase, 31.0 kDa; trypsin inhibitor, 20.1 kDa; and hen egg-white lysozyme, 14.4 kDa.
Tris mops sds running buffer
Manufactured by GenScript
Sourced in United States
Tris-MOPS-SDS running buffer is a pre-formulated solution used in electrophoresis techniques, specifically for the separation of proteins or nucleic acids. It provides the necessary ionic environment and pH conditions required for effective sample migration and resolution during the electrophoresis process.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (3 mentions)
Chemidoc touch, by Bio-Rad (3 mentions)
Western lighting plus ecl detection reagent, by PerkinElmer (2 mentions)
Expressplus page, by GenScript (2 mentions)
Gel doc ez imager, by Bio-Rad (1 mentions)
Biorad mini protean tetra system, by Bio-Rad (1 mentions)
Imagescanner, by GE Healthcare (1 mentions)
Quantity one software, by GE Healthcare (1 mentions)
Surepage, by GenScript (1 mentions)
Instantblue coomassie protein stain, by Abcam (1 mentions)
Broad multi color pre stained protein standard, by GenScript (1 mentions)
Pierce bca protein assay kit 23225, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Er sc 543, by Santa Cruz Biotechnology (1 mentions)
Anti goat sc 2020, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000, by Fujifilm (1 mentions)
Criterion tgx gel, by Bio-Rad (1 mentions)
Criterion xt tris acetate gel, by Bio-Rad (1 mentions)
Trans blot turbo rta transfer kit, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
10 protocols using tris mops sds running buffer
1
SDS-PAGE Analysis of Protein Samples
2
SDS-PAGE Protein Analysis Protocol
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the procedure of Xiong et al. with minor modifications.16 (link) Prior to electrophoresis, a given volume of protein sample was mixed with the SDS-PAGE sample buffer (4% SDS, 20% glycerol, and 0.125 M Tris, pH 6.8), with or without 5% βME, and made up to a final protein concentration of 1.5 mg mL−1. For samples without βME, 0.5 mM N-ethylmaleimide (a thiol blocking agent) was added to prevent disulfide artifacts. All mixtures were heated in boiling water (100 °C) for 5 min. A 12 μL sample aliquot (18 μg of protein) was loaded onto the 4–12% precast gel. The gels were run in Tris-MOPS-SDS running buffer (1 L; GenScript, USA) at 140 V for 1.5 h. Proteins were then stained with Coomassie Brilliant Blue R250 for 0.5 h and destained until the bands were clear. Gel images were captured using an image scanner (GE Healthcare, Little Chalfont, UK), and the band intensities were quantified using Quantity One software (GE Healthcare, Little Chalfont, UK). The relative intensity of each band was calculated from the actual intensity of the measured band relative to the actual intensity of the 130 kDa band in the prestained calibration marker.
3
Proteomic Profiling of Spermatozoa
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The fresh semen was washed three times using phosphate buffered saline (PBS) and centrifuged at 1,800 rpm. Following the manual’s instructions, the spermatozoa pellet was extracted using PRO-PREPTM protein extraction solution (iNtRON Biotechnologi, Korea). The total soluble protein concentration of the sample was measured before analysis by SDS-PAGE. The colorimetric detection and quantification of total protein were conducted using the bicinchoninic acid (BCA) method, employing the Pierce™ BCA Protein Assay Kit 23225 from Thermo Scientific™, USA. SDS-PAGE analysis was performed to determine the protein profile based on MW, which is represented as bands on the gels. Protein separation was carried out using SDS-PAGE using SurePAGE™, Bis-Tris, 10 × 8 cm, 12 wells, 4%–20% gradient gel (M00656; GenScript) (SurePAGE, Genscript Biotech Corp. Hongkong) with Broad Multi Color Pre-Stained Protein Standard (M00624; GenScript) with a MW range of ~5–270 kDa and Tris-MOPS-SDS Running Buffer (M00138; GenScript) with a voltage of 140 V and a current of 75 mA for 55 min. The gel was then stained using InstantBlue® Coomassie Protein Stain (ab119211; abcam). The differential intensity of individual protein bands was assessed by conducting ratio analysis with the aid of ImageJ software [9] .
4
Western Blot Analysis of ER and CDK Signaling
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Protein (50 µg) was resolved on 8% Express-Plus PAGE gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ, USA), transferred to PVDF membranes and incubated at 4°C overnight with primary antibodies: CDK8 (sc-1521, SantaCruz, Santa Cruz, CA, USA), CDK19 (HPA007053, Sigma-Aldrich), ER (sc-543, SantaCruz), phospho-ER Ser118 (sc-101675, SantaCruz) and GAPDH (#5174, Cell Signaling Technology, Danvers, MA, USA) followed by either anti-goat (sc-2020, SantaCruz), anti-rabbit (#31460, ThermoFisher Scientific) or anti-mouse (31430, ThermoFisher Scientific) secondary antibodies. Bands were visualized with Western Lighting Plus ECL detection reagent (Perkin Elmer, Waltham, MA, USA) using ChemiDoc Touch™ (BioRad). Images were analyzed and densitometry performed using ImageLab software (Biorad).
5
Western Blot Analysis of Tau and GFP
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Samples were run on either 4–20% Express Plus PAGE in Tris-MOPS-SDS running buffer (GenScript), 10% Criterion TGX gels (Bio-Rad) in Tris-glycine-SDS running buffer, or 3–8% Criterion XT Tris acetate gels (Bio-Rad) in Tris acetate-SDS running buffer. Proteins were transferred to PVDF membranes (Trans-Blot Turbo RTA transfer kit, Bio-Rad) using the Trans-Blot Turbo transfer system (Bio-Rad) and immunoblotted with the Tau antibody A-10 (1:1000–10,000, mouse, sc-390476, Santa Cruz Biotechnology) or anti-GFP (1:10,000, mouse, MMS-118P, Covance). An alkaline phosphatase-coupled secondary antibody (Vector Laboratories) together with ECF substrate (GE Healthcare Life Sciences) was used for development. The blots were imaged on an ImageQuant LAS-4000 (FUJIFILM Co.). Densitometric quantification of the signals was performed with Image Studio Lite software (LI-COR Biosciences).
6
Western Blot Analysis of STAT1 and pSTAT1-Ser727
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Cells were cultured in 60-mm dishes and treated under different conditions before being lysed in RIPA lysis buffer with protease/phosphatase inhibitor cocktail. The protein concentration of extracts was determined using the DC protein assay (Bio-Rad Laboratories). Protein samples (50 μg) were resolved on 4–12% Express-Plus PAGE gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ, USA), transferred to PVDF membranes and incubated at 4 °C overnight with primary antibodies STAT1 (sc-592, Santa Cruz Biotechnology, Dallas, TX, USA) and pSTAT1-Ser727 (#8826, Cell Signaling Technology, Danvers, MA, USA) followed by anti-rabbit (NA934, GE Healthcare, Chicago, IL, USA) secondary antibodies. Bands were visualized with Western Lighting Plus ECL detection reagent (Perkin Elmer, Waltham, MA, USA) using ChemiDoc Touch™ (Bio-Rad). Images were analyzed using ImageLab (Bio-Rad) and ImageJ (version 1.52p) software for protein signal quantification.
7
Western Blot Analysis of CDK8 and CDK19
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Cells were plated in 60 mm plates at a density of 1 × 106 cells per plate in regular culture media and cultured for 24 h. Then cells were treated with 1 μM senexin B or solvent control (0.1% DMSO, MilliporeSigma, St. Louis, MO, USA) for 3 h before lysing cells in 0.5 mL RIPA (radio immunoprecipitation assay) lysis buffer with 1× protease inhibitor cocktail. The protein concentration of extracts was determined using the DC (detergent-compatible) protein assay (Bio-Rad Laboratories). Protein (50 μg) was resolved on 4–12% Express-Plus polyacrylamide gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ, USA), transferred to the PVDF (polyvinylidene difluoride) membrane, blocked with 5% non-fat milk and incubated with primary antibodies: CDK8 (sc-1521, Santa Cruz Biotechnology, Dallas, TX, USA), CDK19 (HPA007053, MilliporeSigma) and GAPDH (sc-32233, Santa Cruz Biotechnology) followed by either anti-goat (sc-2020, Santa Cruz Biotechnology), anti-rabbit (NA934, GE Healthcare, Chicago, IL, USA) or anti-mouse (NXA931, GE Healthcare) secondary antibodies. Bands were visualized with Western Lighting Plus ECL (enhanced chemiluminescence) detection reagent (Perkin Elmer, Waltham, MA, USA) using ChemiDoc Touch™ (Bio-Rad Laboratories, Hercules, CA, USA). Images were analyzed using ImageLab software (Bio-Rad, Version 5.2.1 build 11).
8
Protein Denaturation and SDS-PAGE Analysis
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Fifteen microliters of purified protein solution was mixed with 5 μl of 4× loading buffer (Life Technologies). The mixed solution was boiled for 10 min for protein denaturation. The processed solution was added to the lane of 12% polyacrylamide gel (GenScript), which was put into tris-Mops-SDS running buffer (GenScript) with an applied 120-V voltage for 60 min and then stained using 0.25% Coomassie Brilliant blue. The SDS image was obtained by the ChemiDoc MP system (Bio-Rad).
9
Cilia Protein Detection by Western Blot
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Isolated cilia were denatured in LDS-sample buffer (Boston BioProducts) for 10 min at 95C. The denatured samples were then loaded onto a precast 4–20% SurePAGE Bis-Tris gel (GenScript) and separated by electrophoresis in Tris-MOPS-SDS running buffer (GenScript) before being transferred to nitrocellulose membranes (Cytiva). Membranes were blocked with 5% skim milk in TBST (Tris-buffered saline with Tween-20), followed by addition of primary antibodies (Key Resources Table ). Antibodies against PKD2-Cterm48 (link) and β-tubulin (Cell Signaling Technology, RRID: AB_2210545) were used at a 1:1000 dilution. Antibodies against PKD2-loop, 48 (link) MST1,19 (link) and SIP19 (link) were used at 1:2000 dilution. Primary antibodies were detected with a horseradish peroxidase-conjugated anti-rabbit antibody (Sigma-Aldrich, RRID: AB_92591) at 1:2000 dilution. After washing in TBST, peroxidase substrate (Cytiva) was applied to the membrane and imaged using a Gel Doc XR+ imaging system and the Image Lab software (Bio-Rad).
10
Oligomeric State Analysis of OsWRKY45-DBD
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Glutaraldehyde crosslinks primary amines and was employed to examine the oligomeric state of OsWRKY45–DBD because there are primary amine groups (Lys) on the surface of OsWRKY45–DBD molecules. In a typical crosslinking reaction, a total volume of 20 μl contained 2 μg protein and 0.04% or 0.08% (v/v) glutaraldehyde (ρ = 0.943 g/ml), rendering the molar ratio of protein:glutaraldehyde are 4:1 or 2:1, respectively. The reaction was carried out at 293 K for 15 min and quenched by 0.1 M Tris (pH 8.0) for 5 min. The products were fractionated on reducing SDS-PAGE with SurePage™ gels (GenScript) and Tris-MOPS-SDS running buffer (GenScript) and visualised by staining with Coomassie Brilliant Blue R250.
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