Forty μg of lung tissue lysate proteins, as determined by a Bradford protein assay were loaded equally onto 10% polyacrylamide gels (Life Technologies Corporation; Carlsbad, CA), and transferred to PVDF membranes (Life Technologies, Corp.). Membranes were blocked for one hour at room temperature in 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST), and then probed with anti-mouse phospho (p)-SHP-1 (Tyr564), p-SHP-2 (Tyr580), p-Akt (Thr308) or SHP-1, SHP-2, Akt antibodies (Cell Signaling Technology, Inc., Danvers, MA) overnight at 4°C in TBST containing 5% bovine serum albumin (BSA). After primary antibody incubation, membranes were washed three times in TBST and rabbit anti-mouse secondary antibody (Cell Signaling Technology, Inc.) was added at a concentration of 1:3000 in TBST with 5% BSA. β-actin was blotted as a loading control. Membranes were developed by chemiluminescence using an Amersham prime ECL plus detection system (GE Healthcare Life Sciences; Pittsburgh, PA) and densitometric analyses were performed as previously reported by our laboratory [21 (link)].
Amersham prime ecl plus detection system
Manufactured by GE Healthcare
Sourced in United States
The Amersham Prime ECL Plus Detection System is a chemiluminescence-based detection system designed for western blotting applications. It is used to detect and visualize proteins that have been separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Rabbit anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Phospho iκbα, by Cell Signaling Technology (1 mentions)
Bradford protein assay kit, by Thermo Fisher Scientific (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Phospho nf κb p65, by Cell Signaling Technology (1 mentions)
2 protocols using amersham prime ecl plus detection system
1
Western Blot Analysis of Lung Tissue
2
Western Blot Analysis of NF-κB Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Livers were carefully excised and homogenized into lysis buffer (Thermo, USA) to yield a homogenate. After centrifugation (12000 g for 10 min) at 4°C, protein concentration was detected by Bradford protein assay kit (Thermo, USA) with bovine serum albumin as standard. Equal amounts of protein extracts separated discontinuously onto 10% polyacrylamide gels (Life Technologies, Carlsbad, CA, USA) and transferred to nitrocellulose membranes (Life Technologies). After blockade of nonspecific binding sites, membranes were incubated with various antibodies against IκBα, phospho-IκBα, NF-κB p65, and phospho-NF-κB p65 (Cell Signaling Technology, Danvers, MA, USA) for 2 h at room temperature. Membranes were developed by chemiluminescence using an Amersham prime ECL Plus detection system (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Signals were densitometrically assessed and normalized to the GAPDH signals [18 (link)].
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