Western blotting was performed to analyze protein expression. Briefly, cell extracts were prepared using the RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 7.4) containing 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and a protease inhibitor (Roche, Cat. 11836170001). The protein concentration was quantified using the Bradford assay reagent (Bio-Rad) according to the manufacturer's instructions. Proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Pall Corporation, Cat. BSP0861). The membranes were blocked with 5% non-fat milk and incubated with the following antibodies at indicated dilutions: anti-ID1 (1:1,000; Biocheck, Cat. BCH-1/195-14-50) and anti-β-actin (1:10,000; Santa Cruz Biotechnology, Cat. sc-47778). Membranes were then incubated with horseradish peroxidase-conjugated anti-IgG secondary antibody (Pierce Biotechnology) and visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Cat. 34580).
Horseradish peroxidase conjugated anti igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Horseradish Peroxidase-Conjugated Anti-IgG Secondary Antibody is a laboratory reagent used to detect and quantify the presence of specific immunoglobulin G (IgG) antibodies in various biological samples. The antibody is conjugated with the enzyme horseradish peroxidase, which can be used as a reporter molecule in immunoassays and other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay reagent, by Bio-Rad (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene fluoride membrane, by Pall Corporation (2 mentions)
A5316, by Merck Group (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Sc 8629, by Santa Cruz Biotechnology (1 mentions)
Sc 490, by Santa Cruz Biotechnology (1 mentions)
Sc 371, by Santa Cruz Biotechnology (1 mentions)
Sc 397, by Santa Cruz Biotechnology (1 mentions)
Ab49261, by Abcam (1 mentions)
Sc 126, by Santa Cruz Biotechnology (1 mentions)
3 protocols using horseradish peroxidase conjugated anti igg secondary antibody
1
Quantification of Protein Expression by Western Blotting
2
Quantifying Stem Cell Markers by Western Blot
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Cell extracts were prepared using RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 7.4) containing 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor (Roche, Switzerland). The protein concentration was quantified using the Bradford assay reagent (Bio-Rad) according to the manufacturer’s instructions. Proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Pall Corporation, USA). Membranes were blocked with 5% non-fat milk and incubated with the following antibodies at the indicated dilutions: anti-OCT3/4 (1:330; sc-8629, Santa Cruz Biotechnology), anti-β-actin (1:10,000; A5316, Sigma-Aldrich). Membranes were then incubated with horseradish peroxidase-conjugated anti-IgG secondary antibody (Pierce Biotechnology, USA) and visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). Quantification of signal intensity of western blot analysis was performed using NIH ImageJ.
3
Western Blot Analysis of Cell Signaling Proteins
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Cell extracts were prepared using RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 7.4) containing 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor (Roche, Basel, Switzerland). Protein concentration was quantified using Bradford assay reagent (Bio-Rad) according to manufacturer instructions. Proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Pall Corporation, Port Washington, NY, USA). Membranes were blocked with 5% non-fat milk and incubated with the following antibodies at the indicated dilutions: anti-p21 (1:500; sc-397), anti-IκBα (1:500; sc-371), anti-p53 (1:500; sc-126, all from Santa Cruz Biotechnology), anti-p-p53 (1:500; 9286, Cell Signaling Technology), anti-ID1 (1:2,000; BCH-1-195-14, Biocheck, Foster City, CA, USA), anti-ID2 (1:500, sc-489, Santa Cruz Biotechnology), anti-ID3 (1:500, sc-490, Santa Cruz Biotechnology), anti-ID4 (1:200; ab49261, Abcam), and anti-β-actin (1:10,000; A5316, Sigma-Aldrich). Membranes were then incubated with a horseradish peroxidase-conjugated anti-IgG secondary antibody (Pierce Biotechnology, Rockford, IL, USA) and visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).
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