Total cellular proteins were extracted by solubilizing the cells in EB buffer (50 mmol/L Hepes pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 5 mmol/L EDTA, 2 mmol/L EGTA; all reagents were from Sigma-Aldrich (St. Louis, MO, USA), in the presence of 1 mmol/L sodium orthovanadate, 100 mmol/L sodium fluoride, and a mixture of protease inhibitors. Extracts were clarified by centrifugation and normalized with the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). Western blot detection was performed with enhanced chemiluminescence system (GE Healthcare, Chicago, IL, USA) and peroxidase conjugated secondary antibodies (Amersham, Little Chalfon, UK). The following primary antibodies were used for Western blotting (all from Cell Signaling Technology, Danvers, MA, USA, except where indicated): anti-phospho-p44/42 ERK (Thr202/Tyr204; 1:1000); anti-p44/42 ERK (1:1000); anti-phospho EGFR (Tyr1068) (1: 1000); anti-EGFR (EnzoLifeSciences, Farmingdale, NY, USA) (1:1000); anti-ferritin light chain (Abcam, Cambridge, UK) (1:1000); anti-Actin (Santa Cruz Biotechnology, Dallas, TX, USA) (1:1000).
Anti phospho p44 42 erk
Manufactured by Enzo Life Sciences
Sourced in United States
The Anti-phospho-p44/42 ERK is a laboratory reagent used for the detection and quantification of phosphorylated forms of the extracellular signal-regulated kinase (ERK) proteins p44 and p42 in biological samples. It serves as a tool for analyzing cell signaling pathways and cellular responses to various stimuli.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence system, by GE Healthcare (2 mentions)
Bca protein assay reagent kit, by Thermo Fisher Scientific (2 mentions)
Anti p44 42 erk, by Enzo Life Sciences (2 mentions)
Anti phospho egfr, by Enzo Life Sciences (2 mentions)
Peroxidase conjugated secondary antibody, by Cytiva (2 mentions)
Anti egfr, by Enzo Life Sciences (1 mentions)
Anti ferritin light chain, by Abcam (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Anti egfr clone13g8, by Enzo Life Sciences (1 mentions)
Ecl system, by Cytiva (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
2 protocols using anti phospho p44 42 erk
1
Extraction and Western Blotting of Cellular Proteins
2
Protein Extraction and Western Blot Analysis
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Total cellular proteins were extracted by solubilizing the cells in cold EB buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA, 2 mM EGTA; all reagents were from Sigma-Aldrich, except for Triton X-100 from Fluka) in the presence of 1 mM sodium orthovanadate, 100 mM sodium fluoride, and a mixture of protease inhibitors (pepstatin, leupeptin, aprotinin, and STI). Extracts were clarified by centrifugation, and protein concentration was determined using BCA protein assay reagent kit (Thermo). Western blot detection was performed with enhanced chemiluminescence system (GE Healthcare) and peroxidase-conjugated secondary antibodies (Amersham). The following primary antibodies were used for western blotting (all from Cell Signaling Technology, except where indicated): antiphospho p44/42 ERK (Thr202/Tyr204); anti-p44/42 ERK; anti-phospho-AKT (Ser473), anti-AKT; anti-phospho EGFR (Tyr1068); anti-EGFR (clone13G8, Enzo Life Sciences); anti-vinculin (Sigma-Aldrich). The following day, after 1 hour of incubation with the appropriate secondary antibody, the signal was developed using the ECL system (Amersham Biosciences).
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