Cells were collected and lysed in RIPR buffer (Beyotime) supplemented with the protease inhibitor cocktail (Roche). Protein concentrations were quantified using the BCA protein assay (Pierce). The cell lysate containing 30 μg total protein was heat‐denatured and subjected to SDS–PAGE, followed by transferring to PVDF membrane. The membrane was incubated with the primary and secondary antibodies and visualized with SuperSignal West Pico Chemiluminescent HRP substrate (Thermo). The anti‐HNRNPC, anti‐PKR, anti‐beta‐actin, and anti‐alpha‐tubulin antibodies were purchased from Abcam. Nuclear and cytoplasmic fraction proteins were separated by Nuclear/Cytosol Fractionation Kit (Biovision, K266‐25). The density quantifications were analyzed by ImageJ software.
Supersignal west pico chemiluminescent hrp substrate
Manufactured by Thermo Fisher Scientific
SuperSignal West Pico Chemiluminescent HRP substrate is a reagent used for the detection of horseradish peroxidase (HRP) in Western blot applications. It is a luminol-based substrate that emits light when oxidized by HRP, allowing for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Abcam (1 mentions)
Anti α tubulin, by Abcam (1 mentions)
Anti pkr, by Abcam (1 mentions)
Anti hnrnpc, by Abcam (1 mentions)
Nuclear cytosol fractionation kit, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti gapdh, by Novus Biologicals (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Image studio lite 5, by LI COR (1 mentions)
Anti p53, by Santa Cruz Biotechnology (1 mentions)
Cpx 130, by Cole-Parmer (1 mentions)
Proteinase inhibitor, by Solarbio (1 mentions)
Ab32197, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Polyvinylidene uoride membrane, by Merck Group (1 mentions)
Ab1791, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab32072, by Abcam (1 mentions)
3 protocols using supersignal west pico chemiluminescent hrp substrate
1
Western Blot Analysis of Nuclear Proteins
2
Protein Expression and Western Blot Analysis
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FL cells were lysed using RIPA buffer with protease and phosphatase inhibitor cocktail (Thermo Scientific), followed by ultrasonication (Cole Parmer CPX-130). Lysates were prepared in LDS sample buffer (Life Technologies) with 10% β-mercaptoethanol and boiled for 5 min for denaturation. Denatured protein lysates were loaded on 4%–15% Tris-HCl PAGE gel (Bio-Rad) for gel electrophoresis separation (50 min at 200 V) and transfer (overnight at 25 V). Transferred membranes were blocked with 5% non-fat dry milk (Carnation, Nestlé) in Tris-buffered saline with Tween 20 and blotted with anti-GAPDH (1:5,000, Novus Biologicals) and anti-p53 (1:500, Santa Cruz), followed by anti-mouse and anti-rabbit IgG horseradish peroxidase conjugates (Promega). Blots were developed on X-ray film after 5-min exposure to the SuperSignal West Pico Chemiluminescent HRP substrate (Thermo Scientific). For quantification, protein band intensities were analyzed using Image Studio Lite 5.2 (LI-COR).
3
Western Blot Analysis of Signaling Proteins
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Cells were lysed using the RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with proteinase inhibitor (Solarbio). Protein concentrations were quanti ed using the BCA protein assay (Pierce). The cell lysate containing 30 µg proteins was heat denatured and subjected to SDS-PAGE, followed by transfer to a polyvinylidene uoride membrane (Millipore, Bedford, MA) by semi-dry blotting. The membranes were blocked with 5% bovine serum album (BSA) at room temperature for 1 h, and then incubated with the primary and secondary antibodies and visualized with the SuperSignal West Pico Chemiluminescent HRP substrate (Thermo). The primary antibodies used in this study were raised against β-catenin (#8480, CST), p-β-catenin (#4176, CST), PRKCA (#2056, CST), GSK-3β (#9315, CST), HBP1 (sc-376831, Santa Cruz), TCF-4 (BS91324, Bioworld ), Cyclin D1 (#2922, CST), AXIN2 (ab32197, Abcam), c-Myc (ab32072, Abcam), β-actin (ab8226, Abcam) and Histone (ab1791, Abcam). Protein levels were calculated relative to β-actin and Histone.
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