The samples were mixed in the ratio of 3:1 with NuPAGE™ LDS sample buffer (4X) (Life Technologies™, Carlsbad, CA, United States). All solutions were then heated at 70°C for 10 min before being added to a precast NuPAGE™ Bis–Tris Gel 10% (Invitrogen, Carlsbad, CA, United States) in the XCell SureLock™ Mini-Cell electrophoresis system (Life Technologies™, Carlsbad, CA, United States). The system was prepared with 1X NuPAGE™ MES SDS Running Buffer (20X) (Life Technologies™, Carlsbad, CA, United States) and 10–250 kDa Precision Plus Protein™ WesternC™ standards (Bio-Rad, Hercules, CA, United States). Electrophoresis was then performed at 100 V for 10 min and subsequently at 140 V for 65 min, and finally, the gel was rinsed with MilliQ water before blotting.
Precision plus protein westernc standard
Manufactured by Bio-Rad
Sourced in United States, Portugal
Precision Plus Protein WesternC Standards are a set of pre-stained protein standards used for the molecular weight determination of proteins in Western blotting applications. The standards contain a mixture of 10 recombinant proteins with molecular weights ranging from 10 to 250 kDa, which are covalently coupled to a blue-purple dye. This allows for the visualization of the protein standards during electrophoresis and protein transfer.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (5 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Precision protein streptactin hrp conjugate, by Bio-Rad (3 mentions)
Mini protean tgx gel, by Bio-Rad (3 mentions)
Bradford protein assay, by Bio-Rad (2 mentions)
Hyperfilm ecl, by GE Healthcare (2 mentions)
A00166, by GenScript (2 mentions)
Anti dsred sc 3909098, by Santa Cruz Biotechnology (2 mentions)
Nzycolour marker 2, by NZYTech (2 mentions)
Bovine serum albumin (bsa), by NZYTech (2 mentions)
Trans blot turbo, by Bio-Rad (2 mentions)
Precast gel, by Bio-Rad (2 mentions)
Molecular imaging software, by Kodak (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
39 protocols using precision plus protein westernc standard
1
SDS-PAGE Protein Separation and Analysis
2
Western Blot Analysis of 4HNE Protein
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The total protein concentration was determined by a bicinchoninic acid (BCA) assay (Thermo Scientific, IL, USA). Samples were boiled for 10 min to induce the denaturation of protein to facilitate the separation of proteins based on size. Protein samples (30 mg) and Precision Plus Protein WesternC standards (Bio-Rad, CA, USA) were resolved on 4 to 15% gradient Mini-Protean TGX stain-free polyacrylamide gels (Bio-Rad) and then transferred onto polyvinylidene difluoride membranes (Merck Millipore, Australia). The blots were blocked with a solution of 5% bovine serum albumin (BSA) in Tris-buffered saline and Tween 20 (T-BST) for 1 h. The blots were then incubated with primary antibody targeting 4HNE (catalog number ab46545; Abcam, Cambridge, MA, USA) in 1% BSA and T-BST overnight, washed, and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (R&D Systems, Minneapolis, MN, USA). The blots were developed using SuperSignal West Femto maximum-sensitivity substrate (Thermo Scientific) and visualized by chemiluminescence (ChemiDoc imaging system; Bio-Rad) (54 (link)).
3
Quantitative Immunoblotting of MMP7
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Immunoprecipitated proteins were subjected to polyacrylamide gel electrophoresis using precast gels (BioRad Laboratories Inc) and a standard preparation Laemmli buffer along with Precision Plus Protein WesternC Standards (BioRad Laboratories Inc). Gel was transferred overnight to nitrocellulose membrane, followed by blocking with 5% nonfat milk, 1% BSA, in PBS for 1 hour. The membrane was then probed with polyclonal rabbit anti-MMP7 (Abcam; Ab38999) primary antibody diluted in PBS-0.05% Tween 20 at 4°C overnight. Polyclonal donkey anti-rabbit IgG-HRP (Abcam) was used as a secondary antibody, along with Precision Protein StrepTactin-HRP Conjugate (BioRad Laboratories Inc) for 1 hour at room temperature. After washing of the membrane, SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific Inc) was used for chemiluminescent detection of probed proteins, visualized with a Kodak Gel Logic 100 system (Eastman Kodak, Rochester, NY). Blot band strengths were quantified using Kodak Molecular Imaging Software (Eastman Kodak) and normalized to baseline uninjured tissue protein levels.
4
Western Blot Analysis of Recombinant Proteins
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Intact cells grown as for the transport assay were washed with 20 mM Tris-HCl, pH 7.5; 10 μl of cells at A600 = 15 were diluted to 40 μl, then sonicated in an ice-cold water bath (Branson 2510) for 5 min 20 μl of the whole cell lysate (equivalent to 7 μg protein as judged by protein assay) was loaded onto SDS-16%PAGE. The gel was transferred onto PVDF membrane by the Trans-Blot Turbo transfer system (Bio-Rad Laboratories) at 1.3 A, 25 V for 20 min. The blocked PVDF membrane by 3% BSA was then reacted with the penta-His antibody (Invitrogen), and washed in 20 mM Tris-HCl, pH 7.5, and 500 mM NaCl for 30 min, and further probed by the secondary Alexa-fluor 680 antibody (Invitrogen) diluted in 3% BSA, 0.1% Tween-20, and 0.01% SDS for 1 h. After washing with 20 mM Tris-HCl, pH 7.5, and 500 mM NaCl for 30 min, the membrane was scanned by LI-COR Odyssey Infrared Image system at 700 nm. Molecular weight markers were indicated (Precision plus protein WesternC standards, Bio-Rad Laboratories).
5
SDS-PAGE Protein Fractionation and Quantification
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For Sup fractions, Laemmli Buffer 2X with beta-mercaptoethanol was added to samples 1:1(v/v), and heated for 5 min in Thermobloc FALC. One part of the sample was further diluted with two parts of Laemmli buffer with beta-mercaptoethanol and centrifuged 3 min. 20 μg of proteins were loaded on Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad) for SDS-PAGE. For Pellet fractions, each sample was heated for 5 min, taken one part and added to nine parts of Laemmli Buffer and centrifuged 3 min. 20 μg of proteins were loaded for SDS-PAGE. Precision Plus Protein™ WesternC Standards, Bio-Safe Coomassie Stain, 10x Tris/Glycine/SDS, Bio-Safe Coomassie Stain purchased from Bio-Rad were used to perform SDS PAGE.
Spectrophotometric measurements for protein assay were carried out with a computer-aided PerkinElmer UV/Vis Lambda 16 spectrophotometer.
Spectrophotometric measurements for protein assay were carried out with a computer-aided PerkinElmer UV/Vis Lambda 16 spectrophotometer.
6
Quantifying PRDM3 Protein Levels in P19 Cells
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The nuclear protein concentrations from the wild-type and PRDM3-deficient P19 cells were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts (20 µg) were separated on 4–15% precast gel (Bio-Rad, Hercules, CA, USA) and transferred to a PVDF membrane (Merck Millipore, Burlington, MA, USA) using a wet transfer. Blots were probed overnight with a primary antibody for PRDM3 (1:1000, Cell Signaling Technology, Danvers, MA, USA) in 5% skim milk in TBS (tris-buffered saline, Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 0.1% tween-20 (Sigma-Aldrich, Saint Louis, MO, USA), followed by incubation with HRP-conjugated anti-rabbit IgG produced in goats (1:5000, Sigma-Aldrich, Saint Louis, MO, USA) in 5% skim milk in TBST for 1 h at RT. Proteins were visualized using the ECL Western Blotting Analysis System (Amersham, Illinois, CA, USA) and ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA). The molecular weight of the proteins was estimated using the Precision Plus Protein WesternC Standards (Bio-Rad, Hercules, CA, USA). Lamin B1 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control for the Western blotting.
7
Ovarian Protein Expression Analysis
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Ovarian tissues stored at different temperatures and for different periods were extracted in EzRIPA Lysis kit (ATTO, Tokyo, Japan) with phosphatase and protease inhibitors, and the samples were separated on 10 to 20% e-PAGEL (ATTO) by SDS-PAGE. Separated proteins were electroblotted onto polyvinylidene difluoride membranes (ATTO). Precision Plus Protein™ Western C Standards (Bio-Rad, Hercules, CA, USA) were used as the molecular weight standard. The blots were blocked with Ez Block Chemi (ATTO) for 1 h (~22 °C) and incubated overnight (4 °C) with the respective primary antibodies: rabbit anti-Caspase 9 (Enzo Life Science, Farmingdale, NY, USA, 1:1000, 46 kDa), rat anti-Chicken-Vasa-Homologue (CVH, generously provided by Prof. Nobuhiko Yamauchi, Kyushu University, 1:3000, 75 kDa) [22 (link),23 (link)] or mouse anti-GAPDH (Proteintech, Rosemont, IL, USA, 1:5000 in TBS-T, 36 kDa). Anti-rabbit, anti-rat or anti-mouse IgG antibodies (Cell Signaling Technology, Danvers, MA, USA 1:2000) conjugated with horseradish peroxidase (HRP) were used as the secondary antibodies, and blots were incubated for 1 h (~22 °C). Immunoreactivity was visualized with ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA).
8
Quantifying CD26 Protein Expression by Western Blot
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First, the amount of protein in the PE was quantified by the Bradford protein assay (Biorad, CA, USA). Samples containing 20 μg of protein were then subjected to 7.5% SDS-PAGE under non-totally reducing (samples with β-mercaptoethanol incubated at 37°C for 15 min) conditions and electroblotted onto PVDF membranes (Millipore, Bedford, MA, USA). Blots were treated with 5% skimmed milk blocking buffer (TBS 1% Tween-20) and were incubated overnight at 4°C. After three washes with TBS-Tween buffer, the membranes were incubated for 1 h at room temperature with anti-human CD26 mAb (clone TP1/16). The TP1/16 anti-CD26 antibody was previously characterized25 (link) in immunoblots of cell lysates under partially denaturing conditions so that it could be compared with the better-known anti-CD26 1F7 mAb, both of which perform considerably better than other antibodies used in several immune techniques26 (link). Goat anti-mouse IgG-HRP conjugated secondary antibody Fc-Specific (Sigma Aldrich) and Precision Plus Protein, Western C Standards (Bio Rad, USA) was used for protein detection before membranes were exposed a few minutes to ECL Prime Western Blotting Reagents substrate solution (GE, Amersham, UK) under appropriate dark room conditions. Results were analysed with ChemiDoc XRS (BioRad) software.
9
SDS-PAGE Analysis of Phage Proteins
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Two-fold serial dilutions of isolated phage protein in 1X Laemmli sample buffer were heated at 95°C for 1 h. A 10-fold dilution of the Precision Plus Protein WesternC Standards (Bio-Rad, Hercules, CA) was diluted in 1X Laemmli sample buffer and heated at 95°C for 10 min. Prepared samples were separated on a 4–20% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad, Hercules, CA) at a constant voltage of 100 V for 45 min. Following separation, the gel was fixed for 10 min in a methanol/acetic acid/water (v/v/v 50:10:40) fixing solution. Protein bands were labeled with a Colloidal Blue Staining Kit (Thermo Fisher Scientific, Waltham, MA) as described in the manufacturer's instructions. Briefly, the fixed gel was incubated in the prepared staining solution (stainer A/stainer B/methanol/water v/v/v/v 20:5:20:55) for 3 h at room temperature with gentle shaking. The staining solution was replaced with distilled water and destained for 11 h at room temperature with gentle shaking. Following destaining, bands were visualized using an EDAS Imaging Station (Eastman Kodak Co., Rochester, NY) equipped with a DC290 digital camera and a white light box. Images were captured and analyzed using Molecular Imaging Software (v. 4.0.3; Eastman Kodak Co., Rochester, NY).
10
Western Blot Analysis of Osteogenic Markers
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The cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, Delaware, CA, USA) containing a 1x Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). The protein content was measured with a protein assay kit (Pierce, Hercules, CA, USA). Protein samples (15 μg), together with a protein marker (Precision Plus Protein Western C Standards; Bio-Rad), were separated on 12% Mini-Protean TGX gels (Bio-Rad, Richmond, CA, USA) for 30 min at 200 V. The separated gels were transferred to a polyvinylidene fluoride (PVDF) membrane for 3 min using the Trans-Blot Turbo Transfer system (Bio-Rad) with Trans-Blot Transfer Packs. Western blots with Runx2), ALP, OSX, and β-actin (ACTB) were processed on the iBind Western System (Life Technologies, Carlsbad, CA, USA) as specified by the antibody manufacturer (anti- Runx2) [Abcam, Tokyo, Japan], anti-ALP [Abcam], anti-Sp7/osterix [OSX; Abcam], and anti- ACTB [Bio-Rad] primary antibodies; and horseradish peroxidase-conjugated anti-mouse secondary antibody [Bio-Rad]). The incubated membranes were developed using an enhanced chemiluminescence system (SignalFire Plus ECL Reagent; Cell Signaling Technology). The band density was quantified using the NIH-Image J software and normalized to that of ACTB on day 0.
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