TAB1 or p38 immunoprecipitates were obtained as above described. Samples were washed twice in lysis buffer and twice in kinase buffer (all from Cell Signaling). Kinase reactions were incubated for 30 min at 30 °C in a the presence or absence of 200 μM ATP (Cell Signaling). In some experiments the ATP competitor p38 inhibitor SB-203580 (10 μM) was added directly to the in vitro kinase reaction, as indicated. Total p38 and phosphorylated p38 (Thr180-Tyr182) levels were detected using the PhosphoTracer ELISA Kit according to the manufacturer’s instructions (Abcam). In vitro kinase assays are shown as proportional to fold increase at 450 nm absorbance emission of triplicate wells ± s.e.m.
Phosphotracer elisa kit
Manufactured by Abcam
Sourced in United Kingdom
The PhosphoTracer ELISA Kit is a quantitative immunoassay designed to measure the levels of phosphorylated proteins in biological samples. It utilizes the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Kinase buffer, by Cell Signaling Technology (2 mentions)
Adenosine triphosphate (atp), by Cell Signaling Technology (2 mentions)
Cell counting kit 8, by Dojindo Laboratories (1 mentions)
Fucoidan, by Merck Group (1 mentions)
Trans membrane protein extraction kit, by Merck Group (1 mentions)
Poly l lysine (pll), by ScienCell (1 mentions)
4 protocols using phosphotracer elisa kit
1
p38 Kinase Assay and Inhibition
2
p38 Kinase Assay and Inhibition
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TAB1 or p38 immunoprecipitates were obtained as above described. Samples were washed twice in lysis buffer and twice in kinase buffer (all from Cell Signaling). Kinase reactions were incubated for 30 min at 30 °C in a the presence or absence of 200 μM ATP (Cell Signaling). In some experiments the ATP competitor p38 inhibitor SB-203580 (10 μM) was added directly to the in vitro kinase reaction, as indicated. Total p38 and phosphorylated p38 (Thr180-Tyr182) levels were detected using the PhosphoTracer ELISA Kit according to the manufacturer’s instructions (Abcam). In vitro kinase assays are shown as proportional to fold increase at 450 nm absorbance emission of triplicate wells ± s.e.m.
3
Aβ-related Compound and Antibody Protocol
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Aβ1–42 was purchased from American Peptide Company (Sunnyvale, CA, USA). XD4 was synthesized by GL Biochem Co., Ltd. (Shanghai, China). The Aβ1–42 and oAβ1–42 kits for Aβ measurement were purchased from IBL Co., Ltd. (Gunma, Japan). Anti-Aβ antibody 6E10 (monoclonal raised against Aβ N-terminal) and 4G8 (monoclonal raised against Aβ17-24) were purchased from Signet Laboratories (Covance, Dedham, MA, USA). Antibodies against SR-A and CD36 were purchased from AbD Serotec (Oxford, UK) and Abcam (Cambridge, UK), respectively. Rabbit anti-His-tag polyclonal antibody was from 4bio Co. (Beijing, China). Fucoidan was obtained from Sigma–Aldrich (St. Louis, MO, USA). Poly-L-lysine was obtained from Sciencell (CA, USA). Transmembrane protein extraction kit was purchased from Merck Millipore (MA, USA). Cell-counting kit (CCK)-8 was purchased from Dojindo (Kumamoto, Japan). Phosphotracer ELISA kit was purchased from Abcam (Cambridge, UK).
4
Quantifying Akt and ERK1/2 Phosphorylation
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The phosphorylation levels of Akt and ERK1/2 was determined by the Phosphotracer ELISA kit (ab185432, Abcam) in a semi-quantitative method. We used RIPA homogenized mouse muscle and the amount of phosphorylated protein was determined according to the manufacturer instructions.
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