The total protein was extracted from cultured cells by cultivating the cultured cells with RIPA lysis buffer (Beyotime Institute of Biotechnology). An enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology) was used to measure protein concentrations. A total of 30 µg protein were separated via SDS-PAGE (10% gel). The separated proteins were then transferred onto polyvinylidene difluoride membranes and blocked with 5% non-fat dried milk at room temperature for 2 h. After overnight incubation at 4°C with primary antibodies targeting PTP4A1 (1:1,000 dilution; cat. no. ab121185) or GAPDH (1:1,000 dilution; cat. no. ab128915) (both from Abcam), horseradish peroxidase (HRP)-conjugated IgG secondary antibody (1:5,000 dilution; cat. no. ab205718; Abcam) was added, and cultivated at room temperature for 1 h. Ultimately, target protein development was achieved using Immobilon® ECL Ultra Western HRP Substrate (EMD Millipore). The densitometry was implemented utilizing Quantity One software (4.62 version; Bio Rad Laboratories, Inc.).
Horseradish peroxidase hrp conjugated igg secondary antibody
Manufactured by Abcam
Sourced in United States
Horseradish peroxidase (HRP)-conjugated IgG secondary antibody is a laboratory reagent used for detection and quantification purposes in various immunoassays and blotting techniques. It consists of an IgG antibody molecule covalently linked to the enzyme horseradish peroxidase. This conjugate enables the amplification and visualization of target antigen signals in immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assays (ELISAs).
Automatically generated - may contain errors
Lab products found in correlation
Immobilon ecl ultra western hrp substrate, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Phospho ikkα β, by Cell Signaling Technology (1 mentions)
Traf2, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Image pro plus version 4, by Media Cybernetics (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Myd88, by Cell Signaling Technology (1 mentions)
Ikkα β, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Gata3, by Cell Signaling Technology (1 mentions)
T bet, by Cell Signaling Technology (1 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
3 protocols using horseradish peroxidase hrp conjugated igg secondary antibody
1
Protein Extraction and Analysis
2
Protein Expression Analysis of Immune Cells
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After 12 or 24 h of treatment, RSC-364 cells and lymphocytes were harvested. Cells were lysed using RIPA Lysis Buffer containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Protein samples (2 mg/ml) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto the polyvinylidene difluoride membranes (Sigma, St. Louis, MO, USA). The membranes were blocked with 5% skim milk and incubated overnight with the following primary antibodies at 4°C: T-bet, GATA-3, RORγt, TRAF2, IKKα/β, phospho-IKKα/β, and NF-κB p50 for lymphocytes; TRAF2, MyD88, IKKα/β, phospho-IKKα/β, NF-κB p50, JAK1, STAT3, and phospho-STAT3 for RSC-364 cells (1 : 1000, Cell Signaling Technology, MA, USA). The membranes were then incubated with horseradish peroxidase- (HRP-) conjugated IgG secondary antibody (1 : 3000, Abcam, Cambridge, MA, USA) at room temperature for 2 h. All immunoreactive proteins were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Three replicates of each experiment were performed. The densitometry values were normalized to GAPDH and quantified using Image-Pro Plus version 4.0 (Media Cybernetics Inc., Rockville, MD, USA).
3
SDS-PAGE and Western Blot Analysis
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Equal amounts (25 µg) of protein from each time point were diluted 1:4 with 5X SDS-PAGE gel loading buffer (0.25M
Tris-HCl, pH 6.8; 15% SDS; 50% glycerol; 25% β-mercaptoethanol; 0.01% bromophenol blue) and incubated at 95 o C for 10 min. Sample separation was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein transfer was carried out using the iBlot Gel Transfer Device (Invitrogen) following the manufacturer instructions. The membrane was blocked for 1 h in blocking buffer (TBS/Tween-20/5% milk) at 20 o C and then incubated overnight at 4 °C with 1:3000 dilution of Lck monoclonal antibody (#MA1-19197, Thermo Fisher Scientific, Rockford, IL, USA). The membrane was washed 3×5 min at 20 ºC in TBS/Tweeen-20 buffer before incubating with the horseradish peroxidase (HRP)-conjugated IgG secondary antibody (#ab99697, Abcam) for 1 h at 20 ºC.
Afterwards, the membrane was washed 3×10 min with TBS/Tween-20 and visualized by using the ECL method.
Tris-HCl, pH 6.8; 15% SDS; 50% glycerol; 25% β-mercaptoethanol; 0.01% bromophenol blue) and incubated at 95 o C for 10 min. Sample separation was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein transfer was carried out using the iBlot Gel Transfer Device (Invitrogen) following the manufacturer instructions. The membrane was blocked for 1 h in blocking buffer (TBS/Tween-20/5% milk) at 20 o C and then incubated overnight at 4 °C with 1:3000 dilution of Lck monoclonal antibody (#MA1-19197, Thermo Fisher Scientific, Rockford, IL, USA). The membrane was washed 3×5 min at 20 ºC in TBS/Tweeen-20 buffer before incubating with the horseradish peroxidase (HRP)-conjugated IgG secondary antibody (#ab99697, Abcam) for 1 h at 20 ºC.
Afterwards, the membrane was washed 3×10 min with TBS/Tween-20 and visualized by using the ECL method.
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