Total protein of cells was extracted using lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) and quantified by Bradford method. 30 µg protein was separated by SDS-PAGE. Samples were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) and incubated overnight at 4°C with antibody against cyclin E (20808; Cell Signaling Technology, Inc., Danvers, MA, USA), matrix metalloproteinase (MMP)2 (1:1,000; cat. no. 4022; Cell Signaling Technology, Inc.), MMP9 (1:1,000; cat. no. 3852; Cell Signaling Technology, Inc.), E-cadherin (24E10) (1:1,000; cat. no. 3195; Cell Signaling Technology, Inc.), N-cadherin (1:600; cat. no. 13116; Cell Signaling Technology, Inc.), p21 (Waf1/Cip1;12D1) (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), p-ERK (1:1,000; cat. no. 9101; Cell Signaling Technology, Inc.), ERK (1:1,000; cat. no. 4695; Cell Signaling Technology, Inc.), β-actin (1:3,000; cat. no. 4967; Cell Signaling Technology, Inc.), GAPDH (1:3,000; cat. no. G5262; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After incubation with peroxidase-coupled anti-mouse/rabbit IgG (1:1,000; Cell Signaling Technology, Inc.) at 37°C for 2 h, bound proteins were visualized using ECL (Pierce; Thermo Fisher Scientific, Inc.) and detected using a DNR BioImaging System (DNR, Jerusalem, Israel). Relative protein levels were quantified using ImageJ software.
Peroxidase coupled anti mouse rabbit igg
Manufactured by Cell Signaling Technology
Sourced in Israel, United States
Peroxidase-coupled anti-mouse/rabbit IgG is a secondary antibody conjugated with horseradish peroxidase. It is used to detect and visualize primary antibodies raised in mouse or rabbit during Western blotting and other immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Mmp 9, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
P21 waf1 cip1 12d1, by Cell Signaling Technology (1 mentions)
Matrix metalloproteinase mmp 2, by Cell Signaling Technology (1 mentions)
E cadherin 24e10, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Glut3, by Cell Signaling Technology (1 mentions)
Antibody against cyclin d1, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Cyclin e, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
P iκb, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
4 protocols using peroxidase coupled anti mouse rabbit igg
1
Protein Extraction and Western Blot Analysis
2
Quantitative Protein Analysis and Western Blot
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Total protein was extracted using lysis buffer (ThermoFisher, Rockford, IL) and Bradford method was used to quantify the protein. When SDS-PAGE assay was performed, 30 g of the protein was separated and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). For primary antibody incubation, cleaved caspase 3, cleaved YAP, Bcl-2, GLUT-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:2000; Cell Signaling Technology, Danvers, MA, USA) were incubated overnight at 4°C. For secondary antibody incubation, peroxidase-coupled anti-mouse/rabbit IgG (1:1000, Cell Signaling Technology, USA) was incubated at 37°C for 2 hrs. The result was visualized using ECL (Thermo, Rockford, IL) and detected using a DNR BioImaging System (DNR, Jerusalem, Israel). The quantification of WB band density was performed by using Photoshop CS6 (Adobe).
3
Quantitative Protein Expression Analysis
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Total proteins from cells were extracted in lysis buffer (Pierce, Rockford, IL, USA) and quantified using the Bradford method. 50 μg protein was separated by SDS-PAGE. Samples were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and incubated overnight at 4 °C with antibody against cyclin D1(1:1000, Cell Signaling Technology, Boston, MA, USA), cyclin E (1:1000, Cell Signaling Technology, Boston, MA, USA), p21(1:1000, Cell Signaling Technology, Boston, MA, USA), CDK4(1:1000, Cell Signaling Technology, Boston, MA, USA), CDK6 (1:1000, Cell Signaling Technology, Boston, MA, USA), MMP2(1:1000, Cell Signaling Technology, Boston, MA, USA), MMP9(1:1000, Cell Signaling Technology, Boston, MA, USA) and JunD (1:1000, Cell Signaling Technology, Boston, MA, USA) and GAPDH (1:1000; Santa Cruz, CA, USA). After incubation with peroxidase-coupled anti-mouse/rabbit IgG (1:1000, Cell Signaling Technology, Boston, MA, USA) at 37 °C for 2 h, bound proteins were visualized using ECL (Pierce, Rockford, IL, USA) and detected using a DNR BioImaging System (DNR, Jerusalem, Israel). Relative protein levels were quantified using ImageJ software.
4
Quantification of Apoptosis Proteins
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Total protein of MCF-7 cells was extracted using lysis buffer (Pierce Biotechnology, Inc., Rockford, IL, USA) and Bradford method was used to quantify the protein. When SDS-PAGE assay was performed, 30 µg of the protein was separated and then transferred to polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA, USA). For primary antibody incubation, cleaved caspase 3, caspase 3, cleaved PARP, PARP, p-p65, p65, p-IκB, IκB, Bcl-2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), and GAPDH (1:2,000; Cell Signaling Technology, Inc.) were incubated overnight at 4°C. For secondary antibody incubation, peroxidase-coupled anti-mouse/rabbit IgG (1:1,000; Cell Signaling Technology, Inc.) was incubated at 37°C for 2 h. Sample protein was visualized using ECL (Pierce Biotechnology, Inc.) and detected using a DNR BioImaging System (DNR Bio-Imaging Systems, Ltd., Jerusalem, Israel). Relative protein levels were quantified using ImageJ software.
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