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PV3320 Certificate of Analysis for Lot 1322684A

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PV3320 Certificate of Analysis for Lot 1322684A Certificate of Analysis MAPK8 (JNK1), Inactive , 100 µg Mitogen-activated Protein Kinase 8 (MAP), Inactive, Histidine-tagged Part Number: PV3320 5781 Van Allen Way Lot Number: 1322684A Carlsbad, CA 92008 Immediate Storage: -80°C Phone: 760.603.7200 Shipping Conditions: dry ice www.thermofisher.com Description: Storage and Handling: Recombinant human full-length protein, inactive, Histidine-tagged, Store at -80°C. At first use, aliquot and store at -80°C to avoid multiple expressed in insect cells. freeze-thaws. If properly stored at -80°C, this product is guaranteed for 6 Specific Activity: months from date of purchase. Approximately 2% activity as compared to the active form of the kinase. Storage Buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.05% Triton® X–100, Can be activated by the upstream kinase MAP2K7, mutant. 2 mM DTT and 50% Glycerol. Concentration: 0.87 mg/mL total protein as measured using the Bradford protein assay with BSA as a standard. Calculated 18,300 nM. Aliases: JNK, JNK1, SAPK1 QUALITY ASSURANCE Activation Test: Gel Information for MAPK8 (JNK1), Inactive The coupled MAPK8 activation assay uses active MAP2K7, mutant to Page Description: The SDS- phosphorylate ATF2 substrate. The assay was setup in two states: 1st PAGE and/or Native PAGE MAPK8 phosphorylation by MAP2K7, mutant without 32P-ATP; and 2nd were run on 4-20% Tris-Glycine ATF2 substrate phosphorylation by activated MAPK8 in the presence of Novex® gels (Catalog #: 32P-ATP. The basal activity of MAPK8 was also assayed in the absence of EC6025BOX). MAP2K7, mutant. Lane 1: Invitrogen™ BenchMark™ Protein Ladder (Catalog #: 10747-012). Dilution Buffer: Lane 2: 1 μg MAPK8 (JNK1), 20 mM Tris (pH 7.5), 0.05% Triton® X−100, 0.1 mg/mL BSA, 2 mM DTT Inactive and 10% Glycerol. Lane 3: 2 μg MAPK8 (JNK1), Assay Conditions: Inactive Both the active and inactive enzymes were diluted in enzyme dilution buffer and the reaction was set up in two stages. In stage one, MAPK8 (JNK1), Lane 4: 5 μg MAPK8 (JNK1), Inactive (40 μg/mL final concentration) was incubated with either Inactive 400 μg/mL final concentration mutant MAP2K7 or with dilution buffer to determine activated and basal MAPK8 (JNK1), Inactive activity Lane 5: 10 μg MAPK8 (JNK1), respectively. The assays were performed in 50 mM Tris (pH 7.5), Inactive ® 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.1% Triton X–100, 250 μM ATP for 30 minutes at 30°C. In stage two, 5 μL of the above reaction mix was Purity: transferred to 25 μL of reaction mix containing 50 mM Tris (pH 7.5), 10 mM 90% as determined by a Coomassie® blue stained SDS-PAGE gel. ® MgCl2, 1 mM EGTA, 2 mM DTT, 0.1% Triton X–100, 200 μM ATP, 0.1 μg/mL ATF2 substrate per reaction and trace [32P]–γ–ATP and Molecular Weight: incubated for 10 minutes at 30°C. 47.6 kDa. Calculated from the protein sequence(s). Mass Spectrometry: MAPK8 (JNK1), Inactive was subjected to proteolytic digest followed by mass spec analysis. The resulting MS/MS data verified MAPK8 (JNK1) identity by comparison against the amino acid sequence(s) of the recombinant protein. For questions, please contact our Technical Support Team N. Am Ph#: 800-955-6288 ext. 40266 Email: [email protected] Printed 24/Apr/2018 Version 1.0 Page 1 Certificate of Analysis Part Number: PV3320, Lot Number: 1322684A Protein sequence alignment with reference sequence(s) GenBank Accession Number: NP_002741.1 1 MSYYHHHHHH DYDIPTTENL YFQGAMGSMS RSKRDNNFYS VEIGDSTFTV LKRYQNLKPI GSGAQGIVCA AYDAILERNV AIKKLSRPFQ NQTHAKRAYR IVGN JNK1, INACTIVE 1 ---------- ---------- --------MS RSKRDNNFYS VEIGDSTFTV LKRYQNLKPI GSGAQGIVCA AYDAILERNV AIKKLSRPFQ NQTHAKRAYR NP_002741.1 101 ELVLMKCVNH KNIIGLLNVF TPQKSLEEFQ DVYIVMELMD ANLCQVIQME LDHERMSYLL YQMLCGIKHL HSAGIIHRDL KPSNIVVKSD CTLKILDFGL 73 ELVLMKCVNH KNIIGLLNVF TPQKSLEEFQ DVYIVMELMD ANLCQVIQME LDHERMSYLL YQMLCGIKHL HSAGIIHRDL KPSNIVVKSD CTLKILDFGL 201 ARTAGTSFMM TPYVVTRYYR APEVILGMGY KENVDLWSVG CIMGEMVCHK ILFPGRDYID QWNKVIEQLG TPCPEFMKKL QPTVRTYVEN RPKYAGYSFE 173 ARTAGTSFMM TPYVVTRYYR APEVILGMGY KENVDLWSVG CIMGEMVCHK ILFPGRDYID QWNKVIEQLG TPCPEFMKKL QPTVRTYVEN RPKYAGYSFE 301 KLFPDVLFPA DSEHNKLKAS QARDLLSKML VIDASKRISV DEALQHPYIN VWYDPSEAEA PPPKIPDKQL DEREHTIEEW KELIYKEVMD LEERTKNGVI 273 KLFPDVLFPA DSEHNKLKAS QARDLLSKML VIDASKRISV DEALQHPYIN VWYDPSEAEA PPPKIPDKQL DEREHTIEEW KELIYKEVMD LEERTKNGVI 401 RGQPSPLAQV QQ. 373 RGQPSPLAQV QQ * highlighted residues denote differences from the reference protein sequence(s). Becky. Baker, QA Engineer III Date: 11/Mar/2013 Novex® is a registered trademark of Life Technologies Corporation. Invitrogen™ is a trademark of Life Technologies Corporation. BenchMark™ is a trademark of Life Technologies Corporation. Coomassie® is a registered trademark of Imperial Chemical Industries. Triton® is a registered trademark of Union Carbide Chemicals and Plastics Co., Inc. For Research Use Only. Not for use in diagnostic procedures. For questions, please contact our Technical Support Team N. Am Ph#: 800-955-6288 ext. 40266 Email: [email protected] Printed 24/Apr/2018 Version 1.0 Page 2.
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