Phosphorylation reactions (20μl) contained ~250ng of purified substrate, 1–2 unit of recombinant PKA catalytic subunit, CK1 or CaMKII (all from NEB), and 0.2mM [γ-32P]ATP (adjusted to 250μCi/μmol, Perkin Elmer) in kinase buffer (buffer for PKA: 50mM Tris-Cl pH7.5, 100mM KCl, 5mM MgCl2, and 2.4mM DTT; buffers for CK1 or CaMKII provided by NEB) supplemented with protease inhibitors. Reactions were incubated at 30°C for 30 min and terminated by addition of 3X SDS loading buffer. Proteins were separated by SDS-PAGE, and gels were dried (Bio-Rad) and exposed to a Phosphor Screen (Molecular Dynamics) for 12 hr. The screen was then analyzed with a Typhoon laser scanner (GE Healthcare).
Camkii
Manufactured by New England Biolabs
Sourced in United States
CaMKII is a serine/threonine-specific protein kinase that is activated by calcium/calmodulin. It is involved in the regulation of various cellular processes, including synaptic plasticity, gene transcription, and metabolism.
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Lab products found in correlation
Pka cα, by New England Biolabs (2 mentions)
Gsk 3β, by New England Biolabs (1 mentions)
Kinase reaction buffer, by New England Biolabs (1 mentions)
Flag m2 agarose beads, by Merck Group (1 mentions)
Calmodulin, by New England Biolabs (1 mentions)
Phosphor screen, by GE Healthcare (1 mentions)
Typhoon laser scanner, by GE Healthcare (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Rolipram, by Abcam (1 mentions)
D ap5, by Hello Bio (1 mentions)
Iem 1460, by Hello Bio (1 mentions)
L 689 560, by Bio-Techne (1 mentions)
5 protocols using camkii
1
Radioactive Kinase Reaction Assay
2
In vitro kinase activity assay
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In vitro kinase reactions were performed by mixing 1 μg of the recombinant active GSK3β, PKA, and Ca2+/calmodulin-dependent protein kinase II (CaMK II) (New England Biolabs, BioLabs, Ipswich, MA, USA), all of which were incubated at room temperature for 30 min under a reaction condition that entailed 20 μL of the kinase activity buffer (50 mM Tris HCl, 10 mM MgCl2, 1 mM dithiothreitol, 10 μM ATP, pH 7.5); the reaction was subsequently stopped by adding 10 μL of SDS-PAGE loading buffer. After denaturation at 95 °C for 1 min, protein phosphorylation was analyzed by immunoblotting with the indicated antibodies.
3
FLAG-CPEB3a Protein Purification and Phosphorylation
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HEK-293 cells expressing recombinant FLAG-CPEB3a protein were harvested 36 h post-transfection by scraping in ice-cold lysis buffer containing 10 mM HEPES pH 7.4, 200 mM NaCl, 2 mM EDTA, 1% Tx-100, Complete Mini protease inhibitors (1 tablet/10 ml) and Phos-Stop phosphatase inhibitors (1 tablet/10 ml). Lysates were sonicated for 10 min in ice-cold sonication bath and spun down (15 min/17.000 rcf). Supernatants were mixed with 50 μl of FLAG M2 agarose beads (Sigma), previously blocked with 3% BSA, and incubated for 2 h at 4°C on a rotator, followed by 5x washing with lysis buffer. CaMKII was preactivated at 30°C for 30 min in kinase reaction buffer (New England Biolabs) supplemented with 1.2 μM calmodulin and 2 mM CaCl2. Beads were mixed with kinase buffer containing 1 mM ATP and PKA-Cα (5000 U, New England Biolabs), pre-activated CaMKII (1000 U, New England Biolabs), or the corresponding buffer without enzyme. Sample was incubated for 30 min at 30°C with rotation and then mixed with 50 μl of NuPage reducing sample buffer and separated by SDS PAGE.
4
Phosphorylation of Obscurin Fragments by Kinases
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For analytical purposes, 0.2 mg/ml obscurin RhoGEF fragments (5–20 μM, depending on the fragment used) were phosphorylated by 200 U PKA (Sigma-Aldrich, cat. no. 539576) or 500 U CaMK II (NEB, cat. no. P6060L) or 25 ng CaMK Id (BioVision, cat. no. 7713–5) or 25 ng MST2 (Millipore, cat. no. 14–524) in kinase reaction buffer (30 mM Hepes pH 7.5, 100 mM NaCl, 2 mM MgCl2, 300 μM ATP, 2 mM DTT [+1mM CaCl2 and 1 μg/ml (approx. 3 μM) Calmodulin (NEB, cat. no. 6060S) for CaMK II/Id]). Samples were incubated for 3 h at 30°C in a PCR cycler and the reaction was stopped by addition of SDS sample buffer. For visualisation of phosphoprotein, 2 μg of obscurin substrate were separated by SDS-PAGE and stained with Pro-QTM Diamond Phosphoprotein Gel Stain as described below. For preparative MST2-phosphorylation of obscurin SH3-DH or DH5667-5899, 500 ng MST2 were added to a reaction volume of 200 μL kinase reaction buffer containing a substrate concentration of 110 μM and the reaction was allowed to proceed for 5 hrs at 25°C, followed by further incubation overnight at 8°C. The phosphorylated protein was buffer exchanged into 40 mM Hepes pH 7.5, 100 mM NaCl and 2 mM DTT.
5
Pharmacological Modulation of Neuronal Signaling
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Drugs were prepared as frozen stock solutions (stored below −20 °C). Compounds were as follows: N,N,H,-Trimethyl-5-[(tricyclo[3.3.1.13,7]dec-1-ylmethyl)amino]-1-pentanaminium bromide hydrobromide (IEM-1460; Hello Bio); 4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one (rolipram; Abcam); 4-[(2 S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester (KN-62; Tocris and Hello Bio); D-AP5 (Hello Bio); L-689,560 (Tocris); a catalytic subunit of protein kinase A (PKA Cα, New England Biolabs); Ca2+/calmodulin-dependent protein kinase II (CaMKII, New England Biolabs).
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