Cell lysates were prepared in ice-cold RIPA buffer (Cell Signaling Technology, Inc.) after MLO-Y4 cell lines were treated with six different Ca2+ concentrations for 24 h. Cell lysates were spun to remove cellular debris by centrifuge at 12×103 g for 10 min at +4°C. Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels. The gels were transferred on PVDF membrane by using a wet transfer system overnight and were labeled to perform immunoblot analysis with PHEX (Thermo Fisher Scientific, Inc., bs-12313R), MEPE (bioss, bs-8689R), DMP1 (biorbyt, orb255063), GAPDH (Cell Signaling Technology, Inc.) and Horseradish peroxidase-labeled antirabbit secondary antibodies (Cell Signaling Technology, Inc.). Proteins were visualized by using Image Studio Lite Ver 4.0 in LI-COR. Protein-content determination was measured by the method described by Bradford.
Horseradish peroxidase labeled anti rabbit secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-labeled anti-rabbit secondary antibody is a laboratory reagent used in immunoassay techniques, such as Western blotting and ELISA, to detect the presence of rabbit primary antibodies. It contains an antibody that specifically binds to rabbit immunoglobulins and is conjugated with the enzyme horseradish peroxidase, which can be used to generate a colorimetric or chemiluminescent signal for visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
Tris glycine gel, by Thermo Fisher Scientific (2 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol kit, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Image studio lite ver 4, by LI COR (1 mentions)
Fusion solo, by Avantor (1 mentions)
Ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
N n dimethylformamide (dmf), by Thermo Fisher Scientific (1 mentions)
Survivin, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Low temperature centrifuge, by Thermo Fisher Scientific (1 mentions)
80 c refrigerator, by Thermo Fisher Scientific (1 mentions)
Tunel kit, by R&D Systems (1 mentions)
7 protocols using horseradish peroxidase labeled anti rabbit secondary antibody
1
Quantification of Osteocyte Markers
2
Protein Expression Analysis of Differentiated Cells
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On day 8 of cell differentiation, whole cell protein lysates from differentiated cells were prepared, resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. Target proteins, including phospho-protein kinase B (P-Akt), Akt, phospho-extracellular signal-regulated kinase (P-ERK), ERK, phospho-c-Jun N-terminal kinase (P-JNK), JNK, phospho-P38 (P-P38), P38, peroxisome proliferator-activated receptor gamma (PPAR-γ), CCAAT/enhancer-binding protein alpha (C/EBP-α), C/EBP-β, glucocorticoid receptor (GR), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were detected using primary antibodies and horseradish peroxidase-labeled anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Target proteins were visualized using ECL Plus Western blotting detection reagents (GE Healthcare, Piscataway, NJ, USA). Protein levels were determined densitometrically using a chemiluminescence system (FUSION Solo, PEQLAB Biotechnologie GmbH, Erlangen, Germany), as previously described [53 ].
3
Cisplatin and LLL12B Cytotoxicity Assay
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Cisplatin was purchased from Sigma and was dissolved in ddH2O at a stock concentration of 5 mM in the dark. LLL12B was synthesized at the University of Florida College of Pharmacy. LLL12B powder was dissolved in sterile dimethyl sulfoxide (DMSO) to make a 20 mM stock solution and stored at − 20 °C. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma, and N, N-dimethylformamide (DMF) was obtained from Fisher. IL-6 and Interferon (INF-γ) were purchased from Cell Signaling. The primary antibodies, phosphorylated STAT3 (Y705), STAT3, P-ERK, ERK, P-AKT, AKT, phosphorylated STAT1 (Y701), STAT1, CyclinD1, Survivin, and GAPDH as well as the horseradish peroxidase-labeled anti-rabbit secondary antibodies were obtained from Cell Signaling.
4
Western Blot Analysis of Cell Signaling
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Cell lysates were separated by SDS-PAGE in 8% Tris-glycine gels (Invitrogen Life Technologies, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane. To determine the expression levels of p53, cyclin-dependent kinase 2 (CDK2), BCL2, BAX and p21, blots were probed with their specific antibodies [diluted with 5% bovine serum albumin (BSA) to 1:1,000]. Membranes were probed with horseradish peroxidase-labeled anti-rabbit secondary antibody (diluted with 5% BSA to 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA). Antibody binding was detected by enhanced chemiluminescence detection kit (Amersham International PLC, Buckinghamshire, UK)
5
Comprehensive Biochemical Analyses of Tissue Samples
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Creatinine (Cr) assay kit (R&D Systems, Minneapolis, MN, USA); blood urea nitrogen (BUN) assay kit (R&D Systems); xanthine oxidase assay kit used to determine the activity of superoxide dismutase (SOD; R&D Systems); TUNEL kit (R&D Systems); dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA), TRIzol kit (Invitrogen, Carlsbad, CA, USA), reverse transcription kit (Invitrogen); rabbit anti-Bax-beta, rabbit anti-Bax and rabbit anti-GAPDH primary antibodies, and horseradish peroxidase labeled anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA, USA); ECL luminescent substrate (Invitrogen); color developing powder (Invitrogen); pipettes (Eppendorf, Hamburg, Germany); PCR instrument (Applied Biosystems, Inc., Foster City, CA, USA); UV imaging system (Biometra GmbH, Göttingen, Germany); electronic balance (Sartorious BP121S; Sartorius AG, Goettingen, Germany); −80°C refrigerator (Thermo Fisher Scientific, Schwerte, Germany); low temperature centrifuge (Thermo Fisher Scientific) and frozen slicers (Leica Microsystems, Wetzlar, Germany). The sources of other related instruments and reagents are described in the relevant section.
6
Western Blot Protein Analysis Protocol
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Cell lysates were separated by SDS-PAGE in 8% Tris-glycine gels (Invitrogen Life Technologies, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane. Blots were probed with specific antibodies [diluted with 5% bovine serum albumin (BSA) to 1 : 1000]. Membranes were probed with horseradish peroxidase-labeled anti-rabbit secondary antibody (diluted with 5% BSA to 1 : 1000; Cell Signaling Technology, Inc., Danvers, MA, USA). Antibody binding was detected by using an enhanced chemiluminescence detection kit (Amersham International PLC, Buckinghamshire, UK).
7
Oxcarbazepine Pharmacokinetics and Molecular Mechanisms
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Oxcarbazepine tablets (Novartis Farma S.p.A, Switzerland); dimethylsulfoxide (DMSO) (Sigma); TRIzol kit (Invitrogen); reverse transcription kit (Invitrogen, USA); ELISA kit (R&D system, USA); electrochemiluminescence (ECL) solution (Invitrogen, USA); rabbit anti-Ppg, rabbit anti-MRP1 and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology); horseradish peroxidase-labeled anti-rabbit secondary antibody (Cell Signaling Technology); polymerase chain reaction (PCR) instrument (Applied Biosystems, USA); ultraviolet imaging system (Biometra, Germany); electronic balance (BP121S, Sartorious, Germany); other relevant equipment and reagents were illustrated in relevant parts.
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