Cells were lysed using radioimmunoprecipitation assay buffer (RIPA: 1 % IgepalCA630, 0.5 % sodium deoxycholate, 0.1 % SDS, 2 mM EDTA) supplemented with protease and phosphatase inhibitor cocktails (Sigma Aldrich, Steinheim, Germany). 50 µg of total protein was separated on 4–12 % Nupage bis–tris gels (Life Technologies, Darmstadt, Germany) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The blots were blocked using either 5 % (w/v) BSA or 5 % (w/v) milk in TBST for 1 h and then probed using primary antibodies against the p65 subunit of Nuclear Factor kappaB (NF-kappaB), Inhibitory Protein I-kappa-B-alpha, Caspase-3, PARP or GAPDH (1:1000, Cell Signaling, Danvers, MA) as well as HRP-coupled secondary antibodies directed against rabbit or mouse IgG, respectively (1:2000; Cell Signaling, Danvers, MA). Detection was performed as previously described [20 (link)]. Cytoplasmic and nuclear proteins were extracted using the NE-PER nuclear and cytoplasmic extraction kit (Thermo-Fischer Scientific, Schwerte, Germany).
Hrp coupled secondary antibodies directed against rabbit or mouse igg
Manufactured by Cell Signaling Technology
HRP-coupled secondary antibodies directed against rabbit or mouse IgG are laboratory reagents used to detect the presence of primary antibodies that bind to rabbit or mouse immunoglobulin G (IgG) in various experimental procedures, such as Western blotting, ELISA, and immunohistochemistry. These secondary antibodies are conjugated with the enzyme horseradish peroxidase (HRP), which can catalyze a colorimetric or chemiluminescent reaction, allowing for the visualization and quantification of the target proteins.
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Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Nf kappab, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using hrp coupled secondary antibodies directed against rabbit or mouse igg
1
Western Blot Analysis of NF-kappaB Signaling
2
Western Blot Analysis of Cytoskeletal Proteins
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Cells were lysed using radioimmunoprecipitation assay buffer (1% Igepal CA630, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) supplemented with protease and phosphatase inhibitor cocktails (Sigma Aldrich). Fifty micrograms of total protein was separated using 4% to 12% Nupage bis-tris gels (Life Technologies, Darmstadt, Germany) and transferred onto PVDF membranes (Millipore, Billerica, MA). The blots were blocked using either 5% (w/v) BSA or 5% (w/v) milk in TBST for 1 hour and then later probed using primary antibodies against CDK5R1p35, CDK5, caldesmon, GAPDH (all used at a dilution of 1:1000; Cell Signaling, Danvers, MA) or phospho-caldesmon (Tyr27) (1:500; Santa Cruz Biotechnology, Dallas, TX) as well as HRP-coupled secondary antibodies directed against rabbit or mouse IgG, respectively (1:2000, Cell Signaling). Detection was performed as previously described [16] (link).
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