G401 cells were washed with 800 µl 1X PBS 3 times for 5 min and lysed with 150 µl radioimmunoprecipitation assay per well. For kidney samples, tissue was homogenized in lysis buffer and sonicated for 3 min. The protein concentration of each sample was determined by bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China). Proteins (50 µg/lane) were segregated using 10% SDS-PAGE gel and immunoblotting was performed using polyclonal antibodies against WT1 (AB10840; 1:2,500; Abcam, Cambridge, UK) and β-tubulin (AB6040; 1:5,000; Abcam). The two antibodies were used at 4°C overnight at 1 mg/ml in PBS with 5% non-fat milk according to the manufacturer's protocol. Then the PVDF membrane was probed with horseradish peroxidase (HRP)-conjugated antibodies (1:4,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Finally, immunoreactivity was detected using Chemiluminescent HRP Substrate reagent (Merck Millipore, Darmstadt, Germany) and the signal was analyzed using Bio-Rad ChemiDoc XRS+ (BioRad Laboratories, Inc., Hercules, CA, USA). β-tubulin was used as an internal control. Western blot analysis was performed in triplicate for each group.
Chemiluminescent hrp substrate reagent
Manufactured by Merck Group
Sourced in Germany, United States
The Chemiluminescent HRP Substrate reagent is a laboratory product used to detect the presence and quantity of an enzyme, specifically horseradish peroxidase (HRP), in a sample. The reagent reacts with HRP to produce a luminescent signal, which can be measured to quantify the amount of HRP present. This product is commonly used in various immunoassay techniques, such as Western blotting and enzyme-linked immunosorbent assays (ELISAs).
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xr, by Bio-Rad (2 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated antibody, by Santa Cruz Biotechnology (1 mentions)
M iggκ bp hrp, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Laemmli buffer, by Merck Group (1 mentions)
Gel doc imaging system, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti t ampk, by Cell Signaling Technology (1 mentions)
Image lab software 6, by Bio-Rad (1 mentions)
Anti pacc, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
5 protocols using chemiluminescent hrp substrate reagent
1
Quantitative Western Blot Analysis of WT1
2
Western Blot Analysis of WT1 and GAPDH
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After the transfected cells grew for 48 h in the 6‐well plate, we washed the cells with 1X PBS three times and lysed them with 100ul 1X RIPA per well. Thereafter, the protein concentration was quantified and 20 μg of proteins from each sample were segregated using 10% SDS‐PAGE gel. The samples were then transferred to PVDF membranes and incubated with primary antibodies (WT1: sc‐7385, Santa Cruz, 1:1000 dilution; GAPDH: sc‐47724, Santa Cruz, 1:1000 dilution) and secondary antibodies (m‐IgGκ BP‐HRP: sc‐516102, Santa Cruz, 1:2000 dilution). Finally, blots were detected with Chemiluminescent HRP Substrate reagent (Merck Millipore, Darmstadt, Germany), and the signal was captured by Bio‐Rad ChemiDoc XRS+ (BioRad Laboratories, Inc., Hercules, CA, USA).
3
Quantifying Cathepsin Proteins in Macrophages
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Macrophages were seeded in 6-well plates at a density of 2 × 106 cells per well. Total proteins were recovered with 200 μl of Laemmli buffer (Sigma-Aldrich). Protein extracts were subjected to electrophoresis in 12% SDS-PAGE gels, transferred to a nitrocellulose membrane and blocked with 0.1% Tween 20, 5% of low fat milk TBS (Tris Buffered Saline). The nitrocellulose membrane was then incubated with primary antibodies specific for human cathepsin S, B and L, and β-tubulin (abcam), overnight at 4 °C. All membranes were washed and incubated with secondary HRP-conjugated antibodies. The bands were visualized with a chemiluminescent HRP-substrate reagent (Merck Millipore) and quantified using ImageJ59 (link).
4
AMPK Phosphorylation Analysis by Western Blot
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Cell lysates were subjected to SDS-PAGE and western blot analysis. Briefly, cells were lysed with protein lysis buffer (RIPA, Thermo) followed by heat denaturation. Five micrograms of whole cell proteins were applied to SDS-PAGE. After electrophoresis, the proteins were transferred onto PVDF membrane, followed by membrane blocking in TBS-T buffer containing 5% non-fat dry milk for 1 hour at room temperature. The membrane was probed with the following different primary antibodies: anti-phosphorylated-AMPK (Thr172) (Cell Signaling), anti-tAMPK (Cell Signaling), anti-pACC, and anti-GAPDH (Santa-Cruz). Then, the membrane was washed and incubated with peroxidase-conjugated secondary antibody and finally visualized using Chemiluminescent HRP Substrate reagent (Millipore MA, USA) in Gel-Doc imaging system (Bio-Rad). Signals from Western blot were quantified by using Image Lab Software 6.0 (Bio-Rad).
5
Protein Analysis of KRAS, P15, P16, and P53
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Cells were lysed with RIPA buffer and total protein was quantified with a BCA kit (Beyotime Institute of Biotechnology, Shanghai, China; cat. no. P0009). Proteins were separated by SDS-PAGE followed by immunoblotting with primary antibodies against KRASG12D (NewEast Biosciences, Malvern, PA, USA; cat. no. 26036); P15 (cat. no. 12877-1-AP) and P16 (cat. no. 10883-1-AP) (both from Proteintech); and P53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat. no. sc-6243). Specific signals were detected with horseradish-peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA) and chemiluminescent HRP substrate reagent (Millipore, Billerica, MA, USA; cat. no. WbKLS0500).
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