Dulbecco’s Modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), and Hanks’ balanced salt solution were obtained from Nissui (Tokyo, Japan). Fetal bovine serum (FBS) and calf serum (CS) were obtained from JRH Bioscience (Tokyo, Japan). Anti-Myc monoclonal antibody (9E10) was purchased from Millipore (Billerica, MA, USA). Anti-FLAG M2 antibody and anti-α-tubulin antibody (B-5-1-2) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-GFP monoclonal antibody (B-2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PI3KAP/XB130 antibody was raised in our laboratory as previously described (12 (link)). Alexa Fluor 488-conjugated anti-mouse IgG antibody was from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-linked anti-mouse IgG antibody and HRP-linked anti-rabbit IgG antibody were purchased from GE Healthcare (Buckinghamshire, UK). Other chemicals were of reagent grade available commercially.
Hrp linked anti rabbit igg antibody
Manufactured by GE Healthcare
Sourced in United Kingdom
The HRP-linked anti-rabbit IgG antibody is a laboratory reagent used for the detection and quantification of rabbit immunoglobulin G (IgG) in various immunoassays. The antibody is conjugated with horseradish peroxidase (HRP), which serves as a reporter molecule, enabling the visualization and measurement of target rabbit IgG.
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Lab products found in correlation
Horseradish peroxidase hrp linked anti mouse igg antibody, by GE Healthcare (2 mentions)
Dulbecco s modified eagle s medium, by Nissui Pharmaceutical (1 mentions)
Alexa fluor 488 conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin antibody b 5 1 2, by Merck Group (1 mentions)
Anti gfp monoclonal antibody b 2, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Nissui Pharmaceutical (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Hrp linked anti mouse igg antibody, by GE Healthcare (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Trans blot turbo mini pvdf membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Mouse anti beta actin antibody, by Merck Group (1 mentions)
Rabbit anti smad2 antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
5 protocols using hrp linked anti rabbit igg antibody
1
Cell Culture Reagents and Antibodies
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted using the RIPA buffer. The extraction and isolation of nuclear and cytoplasmic proteins were performed with the Cytoplasmic and Nuclear Protein Extraction kit (cat no. 78833, Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The protein concentration was determined using the bicinchoninic acid assay (Pierce, Thermo Fisher Scientific Inc.), following centrifugation of the protein fraction at 10,000 × g for at 4°C for 15 min. Proteins (20-50 μg) were separated by 12% SDS-PAGE (w/v) and transferred onto PVDF membranes (EMD Millipore). The membranes were then blocked with 1% BSA for 2 h at room temperature, and incubated with the following primary antibodies overnight at 4°C: TAK1 (cat no. 5206, 1:1,000), Bax (cat no. 5023, 1:1,000), Bcl-2 (cat no. 3498, 1:1,000), nuclear p-p65 (cat no. 3036, 1:1,000), p-IκB-α (cat no. 2859, 1:1,000), IκB-α (cat no. 4814, 1:1,000), histone H3 (cat no. 9728, 1:1,000) and β-actin (cat no. 4970, 1:1,000). Subsequently, the membranes were incubated with HRP-linked anti-rabbit IgG antibody (cat no. 7074, 1:2,000) and incubated with ECL reagent (GE Healthcare) for the detection of protein expression. All antibodies were obtained from Cell Signaling Technology, Inc. The gray value of the analyzed proteins was determined using the ImageJ software (version 1.46; Rawak Software Inc.).
3
Affinity Purification of S-tagged Proteins
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293T and Vero cells were cultured in DMEM supplemented with 10% fetal calf serum. Western blots were performed as described previously39 (link), 41 (link). For pull-down assays, transfected 293T cells were lysed by HNTG buffer (20 mM HEPES (pH 7.9), 0.18 M NaCl, 0.1% NP-40, 0.1 mM EDTA, 10% Glycerol)16 (link) with protease inhibitors and sonicated. The cell extracts were subjected to affinity purification using S-protein-immobilized beads (Novagen, MA, USA), and purified proteins (containing 2xS-tagged ORF34 or mutants) were subjected to western blotting. Anti-Myc (9E10) (Calbiochem, MA, USA), Anti-S-probe (K-14) (Santa-Cruz, CA, USA), Anti-FLAG (M2) (Sigma-aldrich) were used as the primary antibodies. HRP linked anti-mouse IgG antibody (GE healthcare UK Ltd., Buckinghamshire, UK) or HRP linked anti-rabbit IgG antibody (GE healthcare UK Ltd.) was used as the secondary antibody.
4
Quantitative Analysis of HO-1 Protein
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The detection of HO-1 protein was carried out following the previous report.(8 ) Kidney was lysed in RIPA buffer (Wako Pure Chemical Industries, Osaka, Japan) containing 1% Halt Protease Inhibitor Cocktail. This lysate was fractionated by 4–15% gradient SDS-PAGE, and electrotransferred onto Trans-Blot Turbo Mini PVDF membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were incubated with primary antibodies: anti-HO-1 antibody (kindly provided from Dr. Shigeru Taketani, Department of Biotechnology, Kyoto Institute of Technology), anti-GAPDH antibody (Enogene Biotech, New York, NY). Following incubation with HRP-linked anti-rabbit IgG antibody (GE healthcare, Princeton, NJ), HO-1 and GAPDH were detected by using Immuno Star LD (292-69903, Wako Pure Chemical Industries, Osaka, Japan) and quantified by ChemiDoc MP system (Bio-Rad Laboratories, Hercules, CA).
5
Western Blot Analysis of TGFBR1 and Smad2
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Cells were lysed with RIPA buffer (Millipore, Billerica, MA, United States) that contained phosphatase and proteinase inhibitors. The protein concentration of the lysates was measured by the Bradford assay (Bio-Rad, Hercules, CA, United States). Aliquots of lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to western blot analysis. Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States). Secondary antibodies were a horseradish peroxidase (HRP)-linked anti-mouse IgG antibody (1:10,000, GE Healthcare, Piscataway, NJ, United States) and HRP-linked anti-rabbit IgG antibody (1:10,000, GE Healthcare). Immunoreactive proteins were detected by SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, United States) with an ImageQuant LAS4,000 (GE Healthcare).
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