Cell lysates were harvested in MPER buffer containing phosphatase and protease inhibitors. Approximately 10–25 µg of protein was separated on polyacrylamide SDS-page gels and then transferred to PVDF membranes, as previously described (13 ). The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA). The secondary antibody used in this study was the rabbit horseradish peroxidase-conjugated (sc-2004; Santa Cruz, Dallas, TX, USA).
β actin hrp sc 47778 hrp
Manufactured by Santa Cruz Biotechnology
Sourced in United States
β-actin HRP (sc-47778 HRP) is a horseradish peroxidase (HRP)-conjugated antibody that recognizes the β-actin protein. β-actin is a cytoskeletal protein found in all eukaryotic cells and is commonly used as a loading control in western blotting and immunoprecipitation experiments.
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2 protocols using β actin hrp sc 47778 hrp
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Western Blot Analysis of Protein Targets
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Immunoblot Analysis of Apoptosis Markers
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Organoids in matrigel were mechanically disrupted and samples were denatured in Laemmli buffer, boiled at 95 °C for 10 min, separated by SDS-PAGE and transferred to nitrocellulose membranes. PBS supplemented with 0.05% Tween-20 (v/v) and 3% nonfat dry milk (w/v) was used for blocking and washing of membranes. Immunoblots were incubated overnight with primary antibodies against full-length and cleaved caspase-3 (9662 S and 9664 S, 1:1000, Cell Signaling), full-length and cleaved caspase-7 (9492 S and 9491 S, 1:1000, Cell Signaling), PARP (9532 S, 1:1000, Cell Signaling), Pyrin (ab195975, 1:1000, Abcam) or tubulin (ab6046, 1:2000, Abcam), followed by HRP-conjugated secondary antibody raised against rabbit (111-035-144, 1:5000, Jackson ImmunoResearch Laboratories). For some loading controls, β-Actin-HRP (sc-47778 HRP, 1:10,000, Santa Cruz) antibody was used. All proteins were detected by enhanced chemiluminescence (Thermo Scientific). Western blot uncropped scans are available in the Supplementary Information.
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