Protein lysates of all cells were extracted with RIPA buffer (Wako Pure Chemical Industries Ltd, Osaka, Japan) including Complete Protease Inhibitor Cocktail and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Ten μg of the protein lysates were loaded onto Mini-PROTEIN TGXTM gel (Bio-Rad Laboratories). After electrophoresis, proteins were transferred to PVDF membranes using the Trans-Blot TurboTM Transfer System Transfer Pack (Bio-Rad Laboratories). Subsequently, the membranes were blocked for 1 h in 4% BSA in TBS with 0.5% Tween-20 (PBS-T) and incubated overnight at 4 °C with primary antibody in TBS-T with 4% BSA. Antibodies concentrations in this study were 1/2500 for rabbit anti-HtrA2 (Cell Signaling Technology) and 1/5000 for HRP-conjugated β-actin (Cell Signaling Technology). After washing three times with TBS-T, membranes were incubated using HRP-conjugated anti-rabbit secondary antibody (Cell Signaling Technology) for 1 h at room temperature. After washing three times with TBS-T, they were visualised using the ECL detection system (GE Healthcare UK Ltd, England, UK). Pictures were taken using a LAS-3000 (Fujifilm, Minato, Tokyo). Protein expression was determined densitometrically and normalised against β-actin expression using the Multi Gauge version 3.1 software (Fujifilm).
Hrp conjugated β actin
Manufactured by Cell Signaling Technology
HRP-conjugated β-actin is a laboratory product used for the detection and quantification of β-actin, a ubiquitous cytoskeletal protein, in various biological samples. The product consists of β-actin antibodies conjugated with horseradish peroxidase (HRP), enabling the visualization and measurement of β-actin levels through colorimetric or chemiluminescent assays.
Automatically generated - may contain errors
Lab products found in correlation
Miniprotein tgxtm gel, by Bio-Rad (1 mentions)
Multi gauge version 3, by Fujifilm (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Ecl plus developing system, by Cytiva (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
3 protocols using hrp conjugated β actin
1
Western Blot Analysis of HtrA2 Protein
2
Comprehensive Western Blot Analysis
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Cell lysates were made with radioimmunoprecipitation assay buffer (RIPA) and western blotting was performed. Membranes were probed with antibodies for CK2α 1:5000 (Cell Signaling, 2656), phosphorylated CK2 substrate 1:10,000 (Cell Signaling, 8738), PARP 1:5000 (Cell Signaling, 9542), β-catenin 1:10,000 (Cell Signaling, 9562), HRP conjugated β-Actin 1:50,000 (Cell Signaling, 5125), or TCF8 (Cell Signaling, 3396). Horseradish peroxidase-conjugated secondary antibodies (Jackson Labs) were incubated for 1 h at room temperature. Blot development was performed with ECL Plus developing system (Amersham Biosciences).
3
Lung Tissue Protein Extraction and Quantification
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As we have previously described9 (link),14 (link),39 (link), lung tissues were perfused with PBS to remove blood and homogenized using RIPA buffer (Sigma) supplemented with protease and phosphatase inhibitor cocktails (Calbiochem). Cells were washed with PBS and protein was extracted using the same modified RIPA buffer solution. Proteins (10–25 μg) were separated on Mini-Protean TGX precast gels (Bio-Rad), transferred to nitrocellulose membranes, and blocked in 5% nonfat milk. Membranes were then probed with primary and secondary antibodies and bands visualized by ECL (Pierce) per manufacturer’s instructions. Densitometry was used to quantify protein levels using ImageJ software, and expression levels were normalized to β-actin. The primary antibodies used were NAMPT, and HRP-conjugated β-actin (Cell Signaling Technology). An anti-rabbit IgG HRP-linked secondary antibody was used (Cell Signaling Technology).
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