P2645

Manufactured by Merck Group

The P2645 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of the P2645 is to provide a controlled environment for various scientific experiments and procedures. Further details about its intended use or specific applications are not available.

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Lab products found in correlation

Axopatch 200b, by Molecular Devices (2 mentions) Mgatp, by Merck Group (1 mentions) Kinase glo reagent, by Promega (1 mentions) Amp pnp, by Merck Group (1 mentions) Rp camps, by Biolog (1 mentions) Kinase glo luciferase reagent, by Promega (1 mentions) Cytation 5 spectrophotometer, by Agilent Technologies (1 mentions) Pclamp 11, by Molecular Devices (1 mentions) Digidata 1550b, by Molecular Devices (1 mentions) A7906, by Merck Group (1 mentions) Micro bio spin p 6 column, by Bio-Rad (1 mentions) Pclamp 9, by Molecular Devices (1 mentions) Digidata 1322a, by Molecular Devices (1 mentions)

Spelling variants of this product

P2645-400 (2 citations)

9 protocols using p2645

Patch-Clamp Electrophysiology with PKA

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For all patch-clamp experiments, pipette solution contained (mM): 140 NMDG-Cl (N-methyl-D-glucamine-chloride), 5 CaCl₂, 2 MgCl₂, and 10 HEPES, pH 7.4 with NMDG. Before patch excision, cells were perfused with a bath solution containing (mM): 145 NaCl, 2 MgCl₂, 5 KCl, 1 CaCl₂, 5 glucose, 5 HEPES, and 20 sucrose, pH 7.4 with NaOH. The bath solution was switched to a standard perfusion solution containing (mM): 150 NMDG-Cl, 2 MgCl₂, 10 EGTA, 8 Tris, and 10 HEPES, pH 7.4 with NMDG, in which the patch was excised to an inside-out configuration.
Purified PKA catalytic subunit was purchased from Sigma-Aldrich (P-2645). MgATP (Sigma-Aldrich) was stored as a 500 mM stock at −20°C. The [MgATP] used in this study was 2 mM unless otherwise indicated. P-ATP [N⁶-(2-phenylethyl)-adenosine-5’-O-triphosphate] was custom-synthesized by Biolog Life Science Institute (Bremen, Germany) and stored as a 10 mM stock at −80°C. All chemicals were diluted to the concentrations indicated in each figure with the standard perfusion solution (pH adjusted to 7.4 with NMDG).

Yeh H.I., Yu Y.C., Kuo P.L., Tsai C.K., Huang H.T, & Hwang T.C. (2021). FUNCTIONAL STABILITY OF CFTR DEPENDS ON TIGHT BINDING OF ATP AT ITS DEGENERATE ATP-BINDING SITE. The Journal of physiology, 599(20), 4625-4642.

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Protein Kinase A: Purification and Assay

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Rp-cAMPS (>99% purity) was purchased from Biolog, while cAMP (>98.5% purity) was purchased from Sigma-Aldrich and S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate (MTSL) was purchased from Toronto Research Chemicals. PKA R1a (91 to 379, 33 kDa), (119 to 379, 29 kDa), and (91 to 244, 17 kDa) constructs were expressed and purified according to previously published protocols (38 (link), 39 (link)). PKA C-subunit (40 kDa) was expressed (38 (link)) or purchased (P2645, Sigma-Aldrich). AMP-PNP was purchased from Sigma-Aldrich. The kinase substrates, PKS (GRTGRRNSI) and PKS2 (GRTGRANSI), were synthesized and purchased from GenScript. The Kinase-Glo reagents were purchased from Promega.

Byun J.A., Akimoto M., VanSchouwen B., Lazarou T.S., Taylor S.S, & Melacini G. (2020). Allosteric pluripotency as revealed by protein kinase A. Science Advances, 6(25), eabb1250.

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Phosphorylation of Histone Proteins

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The His₆-TTD and His₆-TTD-PHD proteins (30 µM) were treated with PKA (P2645, Sigma) at 0.5 U/µl at 30 °C for 1 h in the reaction buffer (50 mM Tris, pH 7.5, 10 mM MgCl₂, 0.02% Triton-X-100, 1 mM ATP, 1 mM DTT). The untreated sample was incubated at 30 °C for 1 h in same buffer condition without the addition of PKA. After PKA treatment, the protein was used for TR-FRET assay as described above. For identification of phosphorylation site(s) on by LC–MS/MS, His₆-TTD-PHD (176.7 uM) was treated with PKA at 2U/µl at 30 °C for 1.5 h in the reaction buffer (50 mM Tris, pH 7.5, 10 mM MgCl₂, 1 mM ATP, 1 mM DTT) with a total volume of 200 µL. For LC–MS/MS, the untreated His₆-TTD-PHD was incubated at 30 °C for 1.5 h in same buffer condition without the addition of PKA. The PKA-treated and non-treated His₆-TTD-PHD for LC–MS/MS were tested in same TR-FRET assay as previously described and despite the different reaction condition, similar higher sensitivity to 2,4-lutidine inhibition was observed for PKA-treated His₆-TTD-PHD (Supplemental Fig. S9).

Chang L., Campbell J., Raji I.O., Guduru S.K., Kandel P., Nguyen M., Liu S., Tran K., Venugopal N.K., Taylor B.C., Holt M.V., Young N.L., Samuel E.L., Jain P., Santini C., Sankaran B., MacKenzie K.R, & Young D.W. (2021). Discovery of small molecules targeting the tandem tudor domain of the epigenetic factor UHRF1 using fragment-based ligand discovery. Scientific Reports, 11, 1121.

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Phosphorylation of GFAT-1 by PKA

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PKA catalytic subunit from bovine heart (PKA, EC 2.7.11.11, Sigma-Aldrich P2645) was reconstituted in bi-distilled water containing 6 mg/ml DTT at a concentration of 50 µg/ml. Purified GFAT-1 variants were phosphorylated in an assay mixture containing 10 mM MgCl₂, 2 mM Na-ATP, and 20 U PKA in 100 µl GFAT-1 SEC buffer for 30 min at 30 °C. The samples were stored frozen. For proteomic analysis, 5 µg GFAT-1 was alkylated by 5 mM chloroacetamide, reduced with 1 mM TCEP, and digested by 0.1 µg Lys-C endoproteinase (MS Grade, Thermo Fisher Scientific) in 50 mM Tris/HCl, pH 8.3 overnight at 37 °C. The digest was acidified by addition of formic acid (end concentration 0.1%) and the resulting peptides were purified using C18 STAGE tips^{70 (link)}.

Ruegenberg S., Mayr F.A., Atanassov I., Baumann U, & Denzel M.S. (2021). Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1. Nature Communications, 12, 2176.

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Kinetic Analysis of PKA Phosphorylation

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Phosphorylation in the presence of 10 nM PKA C-subunit (P2645, Sigma-Aldrich) and increasing concentration of PKS (GRTGRRNSI; 0 to 30 μM; GenScript) or PKS2 (GRTGRANSI; 0 to 300 μM; GenScript) was allowed to progress for 25 min in 50 μl of the assay buffer [40 mM tris (pH 7.5), 20 mM MgCl₂, 10 μM adenosine 5′-triphosphate (ATP), and bovine serum albumin (BSA; 0.1 mg/ml)]. To reach the final reacting concentrations stated above, we diluted the stock concentrations of PKA C-subunit and the substrates 10-fold. The kinase-catalyzed reaction was terminated by adding 50 μl of the Kinase-Glo Luciferase Reagent (Promega) and was incubated for 10 min at room temperature before measuring the luminescence with a BioTek Cytation 5 spectrophotometer in triplicate. The Michaelis-Menten constant (K_m) was determined through a nonlinear fitting of the V_o versus [S] plot using the Michaelis-Menten equation (V_o = V_o.max [S]/(K_m + [S]).

Byun J.A., Akimoto M., VanSchouwen B., Lazarou T.S., Taylor S.S, & Melacini G. (2020). Allosteric pluripotency as revealed by protein kinase A. Science Advances, 6(25), eabb1250.

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Patch-Clamp Analysis of CFTR Gating

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The patch pipette solution contained 138 mM NMDG, 2 mM MgCl₂, and 5 mM HEPES, pH 7.4 with HCl. The bath solution contained 138 mM NMDG, 2 mM MgCl₂, 5 mM HEPES, and 0.5 mM EGTA, pH 7.1 with HCl. Following excision into the inside-out configuration patches (each from a different cell to enhance biological variability) were moved into a flow chamber in which the composition of the continuously flowing bath solution could be exchanged with a time constant of <100 ms using electronic valves (ALA-VM8, Ala Scientific Instruments). CFTR channel gating was studied at 25°C, in the presence of 2 mM MgATP (A9187-1G, Sigma-Aldrich), following activation by ∼1 min exposure to 300 nM bovine PKA catalytic subunit (P2645, Sigma-Aldrich; activation time constants were <20 s for each construct). Macroscopic currents were recorded at −20/−40/−80 mV and microscopic currents at −80/−120 mV membrane potential. Currents were amplified and low-pass filtered at 2 kHz (Axopatch 200B; Molecular Devices), digitized at a sampling rate of 10 kHz (Digidata 1550B; Molecular Devices), and recorded to disk (Pclamp 11; Molecular Devices).

Simon M.A, & Csanády L. (2023). Optimization of CFTR gating through the evolution of its extracellular loops. The Journal of General Physiology, 155(4), e202213264.

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Fluorescent Labeling of Myofibrils

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Prior to imaging, the chamber was washed three times with 100 μl wash buffer and incubated for 30 min. Myofibrils were introduced through one drilled hole and allowed to attach to the surface for 30 min. To prevent nonspecific binding of subsequent reagents to the coverslip surface, 100 μl of 10 mg/ml BSA (A7906; Sigma-Aldrich) was washed into the chamber for 2 min followed by 100 μl wash buffer. Z-discs were labeled in situ with preconjugated (1 h) 200 nM Alexa-488 goat anti-mouse IgG (A11001; Thermo Fisher Scientific) and 200 nM anti-α-actinin mouse antibody (A7811; Sigma-Aldrich) for 1 h prior to washing with 100 μl wash buffer. Finally, 10 μl imaging buffer (1 mg/ml BSA, 5 nM phosphoenolpyruvate [PEP], 1 μl pyruvate kinase [PK], 6 mM Mg-acetate, 10 mM EGTA, 54 mM Na-acetate, 18 mM Na₂SO₄, 10 mM MOPS, 1 mM DTT, 5 nM Cy3-ATP, and 5 mM ATP, pH 7.4) was washed into the chamber and equilibrated for 10 min prior to imaging. Cy3-ATP was synthesized (Toseland and Webb, 2011 (link)) and provided by Dr. C.P. Toseland (University of Sheffield, Sheffield, UK).
When used, DMSO-solubilized mava (myk-461) was added into the imaging buffer at a final concentration of 30 µM and final DMSO < 1%. cAMP-dependent protein kinase (PKA; P2645; Sigma-Aldrich) was incubated with myofibrils for 1 h before washing into the imaging chamber.

Pilagov M., Heling L.W., Walklate J., Geeves M.A, & Kad N.M. (2022). Single-molecule imaging reveals how mavacamten and PKA modulate ATP turnover in skeletal muscle myofibrils. The Journal of General Physiology, 155(1), e202213087.

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Etv1 Phosphorylation by Protein Kinase A

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Phosphorylation of Etv1 Ser-334 by protein kinase A was performed in vitro using 2.5 units of bovine heart PKA catalytic subunit (P2645, Sigma) with 40 μg of Etv1, in modified buffer B (20 mm HEPES, pH 7.5, 500 mm NaCl, 5% glycerol, 1 mm ATP, 10 mm MgCl₂, and 5 mm dithiothreitol). Reactions were performed for 4 h at room temperature and quenched by the addition of 50 mm EDTA, followed by buffer exchange into buffer B with Micro Bio-Spin® P-6 columns (Bio-Rad) and confirmation of phosphorylation by ESI-TOF mass spectrometry of the intact protein.

Cooper C.D., Newman J.A., Aitkenhead H., Allerston C.K, & Gileadi O. (2015). Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION. The Journal of Biological Chemistry, 290(22), 13692-13709.

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Electrophysiological Characterization of Ion Channels

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Patch pipette solution contained (in mM): 136 N-Methyl-d-glucamine (NMDG) chloride, 2 MgCl₂, 5 Hepes, pH = 7.4 with NMDG. Bath solution contained (in mM): 134 NMDG-Cl, 2 MgCl₂, 5 Hepes, 0.5 EGTA, pH = 7.1 with NMDG. MgATP (2 mM) and 2–50 mM P_i (KH₂PO₄) (Sigma-Aldrich) were added from a 400-mM and a 1 M aqueous stock solution, respectively (pH = 7.1 with NMDG). P-ATP (10 μM) was diluted from a 10 mM aqueous stock (Biolog Life Science Institute, purity 99.6%, with 0.4% P-ADP as the only contaminant identifiable by HPLC). The catalytic subunit of bovine PKA (Sigma-Aldrich P2645) was diluted from an ∼18 μM aqueous stock supplemented with 100 μM dithiothreitol (DTT). The continuously flowing bath solution could be exchanged with a time constant of ∼20 ms using electronic valves (ALA-VM8, ALA Scientific Instruments). Experiments were done at 25 °C; membrane potential was −40 mV. Currents were recorded at a bandwidth of 2 kHz (Axopatch 200B, Molecular Devices), digitized at 10 kHz (Digidata 1322A, Molecular Devices), and recorded to disk (pCLAMP 9, Molecular Devices).

Mihályi C., Iordanov I., Töröcsik B, & Csanády L. (2020). Simple binding of protein kinase A prior to phosphorylation allows CFTR anion channels to be opened by nucleotides. Proceedings of the National Academy of Sciences of the United States of America, 117(35), 21740-21746.

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