P44 42 map kinase assay kit

Manufactured by Cell Signaling Technology

The P44/42 MAP kinase assay kit is a laboratory tool designed to detect and measure the activation of the p44/42 mitogen-activated protein (MAP) kinase pathway in cellular samples. The kit provides the necessary components to perform the assay, including antibodies and reagents for signal detection.

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Lab products found in correlation

Pgl3 basic vector, by Promega (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Cnbr sepharose 4b, by Cytiva (1 mentions)

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P44/42 MAP kinase (5 citations)

2 protocols using p44 42 map kinase assay kit

Characterization of Elk-1 and DNMT1 Regulation

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The antibodies against ERK1, p38, Elk1 and phosphor-ERK were obtained from Santa Cruz Biotechnology. The antibody against DNMT1 was from Epitomics. The antibodies against phosphor-Elk1 (Ser383), Akt, and p44/42 MAP kinase assay kit were purchased from Cell Signaling Technology. CNBr-Sepharose 4B were purchased from Amersham Pharmacia Biotech. PD98059 was obtained from Calbiochem.
The coding region of human Elk-1 was cloned by PCR and inserted into pcDNA3.1 vector to construct the eukaryotic expression Elk-1 plasmid pcDNA3.1-Elk1. The 2193 bp dnmt1 promoter fragment was inserted into pGL3-Basic vector (Promega) and the plasmid was designated as pGL3-dnmt1. The full-length coding region of MEK1 with mutations(S218,S222) or MEK2 with mutations(S222,S226) was inserted into GV141 vector by XhoI and KpnI to construct the eukaryotic expression constitutively active MEK1 and MEK2 plasmids, respectively.
Grifolin (2-trans, trans-farnesyl-5-methylresorcinol) was provided by Kunming Institute of Botany, the Chinese Academy of Sciences (purity > 99%, HPLC analysis). Dimethyl sulphoxide (DMSO, Sigma) was used to dissolve grifolin. The final concentration of DMSO in the culture media was kept less than 0.1% v/v which had no significant effect on the cell growth.

Luo X., Yang L., Xiao L., Xia X., Dong X., Zhong J., Liu Y., Li N., Chen L., Li H., Li W., Liu W., Yu X., Chen H., Tang M., Weng X., Yi W., Bode A., Dong Z., Liu J, & Cao Y. (2015). Grifolin directly targets ERK1/2 to epigenetically suppress cancer cell metastasis. Oncotarget, 6(40), 42704-42716.

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Quantitative Analysis of ERK Activation

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The cells were lysed and sonicated in a buffer containing Tris (10 mm, pH 7.5), NaCl (150 mM), EGTA (2 mM), orthovanadate (1 mM), DTT (2 mM) and protease inhibitors: aprotinine (10 μg/ml), leupeptin (10 μg/ml) and phenylemethanesulfonyl fluoride (PMSF) (1 mM) for 30 min at 4°C. Activity was assessed using p44/42 MAP kinase assay kit (Cell Signaling Technology, Inc.). Briefly, the lysates were immunoprecipitated with immobilized phopho-p44/42 MAP kinase monoclonal antibody for 5 h at 4°C and the immune complexes were washed three times with lysis buffer, once with kinase buffer, and resuspended in kinase buffer containing Elk-1 fusion protein. The reactions were incubated for 30 min at 30°C and terinated by the addition of SDS sample buffer and analyzed by immunoblotting with anti-phopho-Elk-1 antibody and ERK1/2 antibody. The antigen antibody complexes were visualized by chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). Real time PCR analysis was performed for quantification of ERK using the Mx3000P (Stratagene, La Jolla, CA, USA).

Seo H.J., Choi S.J, & Lee J.H. (2014). Paraquat Induces Apoptosis through Cytochrome C Release and ERK Activation. Biomolecules & Therapeutics, 22(6), 503-509.

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