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Creatine phosphokinase

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Creatine phosphokinase is a lab equipment product used to measure the activity of the enzyme creatine kinase, which is responsible for the conversion of creatine to phosphocreatine, an important energy storage molecule in the body. The measurement of creatine phosphokinase levels can provide information about the health of certain tissues, such as the heart and skeletal muscles.

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Lab products found in correlation

Creatine phosphate, by Merck Group (18 mentions) Phosphocreatine, by Merck Group (9 mentions) Adenosine triphosphate (atp), by Merck Group (7 mentions) Phosphocreatine disodium, by Merck Group (6 mentions) Creatine phosphate, by MP Biomedicals (4 mentions) Typhoon fla 9500, by GE Healthcare (3 mentions) Adenosine diphosphate (adp), by Merck Group (3 mentions) Dithiothreitol (dtt), by Merck Group (3 mentions) Tris hcl, by Thermo Fisher Scientific (2 mentions) 3 isobutyl 1 methylxanthine, by Enzo Life Sciences (2 mentions) Cgmp eia buffer, by Cayman Chemical (2 mentions) Imark automated plate reader, by Bio-Rad (2 mentions) Mgcl2, by Thermo Fisher Scientific (2 mentions) Sodium nitroprusside, by Merck Group (2 mentions) Hiload 16 60 superdex 200 size exclusion column, by Cytiva (2 mentions) Ni2 nta, by Qiagen (2 mentions) Pd 10 column, by GE Healthcare (2 mentions) Ha ub, by R&D Systems (2 mentions) Pyranose oxidase, by Merck Group (2 mentions) Mineral oil, by Merck Group (2 mentions)

Close spelling variants of this product

20° creatine phosphokinase Creatine phosphokinase (C3755, Creatine phosphokinase type I Creatine

50 protocols using creatine phosphokinase

1

In Vitro Hepatitis B Virus cccDNA Repair

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60 ng of RrcDNA or NeutrAvidin-RrcDNA complex, or 1–5 ng (estimated by quantitative PCR) of deproteinated virion derived rcDNA were incubated with yeast cell extract in reaction buffer consisting of 20 mM Tris-HCl (pH 7.6), 50 mM KCl, 0.1 mM dNTP, 5 mM MgCl2, 1 mM reduced glutathione, 2.6 mM ATP, 26 mM phosphocreatine disodium (Sigma Aldrich, St. Louis, MO), and 6 μg/ml creatine phosphokinase (Sigma Aldrich, St. Louis, MO). The mixture was incubated at 30°C for 60 min before the reaction was stopped by addition of SDS and EDTA to final concentrations of 0.5% (w/vol) and 25 mM, respectively. The solution was subsequently treated with RNase A and proteinase K for 1 hour each. The solution was then extracted by phenol-chloroform (1:1, vol/vol) and repair product precipitated by ethanol and dissolved in TE buffer consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. To monitor cccDNA formation, the repair products were loaded onto a 0.7% (w/vol) agarose gel containing 0.05 μg/ml ethidium bromide and run at 4 V/cm for 2 hours before the gel was visualized on Typhoon™ FLA 9500 (GE Healthcare Lifesciences, Chicago, IL). The intensity of DNA bands was quantified by ImageJ.
For the repair reaction using deproteinated rcDNA (dp-rcDNA), cccDNA was detected by Southern blotting as describe in the Southern blotting section.
Wei L, & Ploss A. (2020). Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation. Nature microbiology, 5(5), 715-726.
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2

LexA Repressor Cleavage Assay

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M13 single-stranded DNA or pBluescript SK(-) double-stranded DNA (18 μM) and RecA (1.0 μM) were first incubated at 37°C for 20–30 min in the standard reaction buffer, except with 5.0-mM MgCl2, containing an ATP-regenerating system. The ATP-regenerating system used in this study consisted of 4.8-mM phosphocreatine and 7.8 units/ml of creatine phosphokinase (Sigma Chemical Co.). LexA cleavage was initiated by the addition of LexA repressor protein (4.7 μM) to the reaction mixture (final, 21 μl), followed by an incubation at 37°C. At the indicated times, 5-μl aliquots were withdrawn. The samples were mixed with 5-μl 2×SDS sample buffer (125 mM Tris-HCl (pH 6.8), 10% 2-mercaptoethanol, 4% sodium dodecyl sulphate (SDS), 10% sucrose, 0.01% bromophonol blue (BPB)) at 0°C, and then heated at 95°C for 5 min to terminate the reaction. The products and unreacted substrates were then separated by electrophoresis through a 15% polyacrylamide gel containing 0.4% SDS. The gel was stained by 0.25% Coomassie Brilliant Blue and photographed. Precision Plus Protein™ All Blue Standards (250, 150, 100, 75, 50, 37, 25, 20, 15 and 10 kDa), purchased from Bio-Rad Laboratories, Inc., were used as protein size markers
Shinohara T., Ikawa S., Iwasaki W., Hiraki T., Hikima T., Mikawa T., Arai N., Kamiya N, & Shibata T. (2015). Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair. Nucleic Acids Research, 43(2), 973-986.
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3

In Vitro Ubiquitination Assay of APC/C

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In vitro ubiquitination assay was performed as described previously (7 (link)), using APC/C immunopurified from mitotic- or G1-synchronized NMuMG, E1KD, or NMuMG-Cdc27 cells using α-Cdc16 antibody (sc-365636; Santa Cruz Biotechnology) and Protein G Sepharose (GE Healthcare Life Sciences). Immunopurified APC/C complexes were incubated with 200 nM recombinant substrate (myc-Cyclin B1 residues 1-102 or full-length GST-Cdh1), 1X ubiquitination reaction buffer (10 mM Tris-Cl pH 7.6, 5 mM MgCl2, 10 mM DTT, 1 mg/ml human recombinant ubiquitin, 10 mM phosphocreatine, 50 μg/ml creatine phosphokinase, 15 mM dATP, 0.15 mg/ml BSA), 250 nM UBE1 (ubiquitin activating enzyme) (Boston Biochem; Cambridge, MA, USA), 1 μM His-UbcH10 (ubiquitin conjugating enzyme), 1 μM His-Ube2S (ubiquitin conjugating enzyme), 1 mg/ml creatine phosphokinase (Sigma-Aldrich; St. Louis, MO, USA), to a volume of 10 μl with water. Reactions were incubated at 37°C for 1 hour with shaking at 1000 rotations per minute. To stop reactions, 5 μl Laemmli sample buffer was added and reactions were vortexed. Myc-Cyclin B1, His-UbcH10, and His-Ube2S were generous gifts of Dr. A. Tipton (Oklahoma Medical Research Foundation; Oklahoma City, OK, USA).
Link L.A., Howley B.V., Hussey G.S, & Howe P.H. (2016). hnRNP E1 Protects Chromosomal Integrity by Translational Regulation of Cdc27. Molecular cancer research : MCR, 14(7), 634-646.
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4

AZ-ODC Degradation Assay in Reticulocyte Lysate

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AZ-ODC proteins at a fixed molar ratio (1:1) were incubated with a rabbit reticulocyte lysate (Promega, Madison, WI, USA) mixture including 40 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM dithiothreitol (DTT), 1.5 mM ATP, 10 mM creatine phosphate (Sigma, St. Louis, MO, USA), and 1.6 mg/mL creatine phosphokinase (Sigma, St Louis, MO, USA) for 2 h at 37 °C. The degradation reaction was stopped after adding the 2× protein sample dye. After the proteins were separated by 13.5% SDS-PAGE, they were transferred to a polyvinylidene difluoride (PVDF) membrane for immunoblotting using anti-ODC (CUSABIO, Houston, TX, USA), anti-AZ (MDBio, Taiwan), and anti-GAPDH (GeneTex, Irvine, CA, USA) antibodies as probes, and the protein bands of ODC, AZ, and GAPDH (internal control) were monitored by ImageQuant™ LAS 4000 (GE Healthcare, Boston, MA, USA). The degradation ratio of ODC at 2 h was quantitated by the program ImageJ when the amount of ODC at 0 h was defined as 1 [53 (link)].
Hsieh J.Y., Liu Y.C., Cheng I.T., Lee C.J., Wang Y.H., Fang Y.S., Liu Y.L., Liu G.Y, & Hung H.C. (2019). Critical Factors in Human Antizymes that Determine the Differential Binding, Inhibition, and Degradation of Human Ornithine Decarboxylase. Biomolecules, 9(12), 864.
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5

Measuring sGC Activity via cGMP Assay

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30 μg of PA protein was measured using the Bradford method. Samples were incubated in a reaction mixture of 50 mM Tris-HCl (pH 7.5, Fisher Scientific), 4 mM MgCl2 (Fisher Scientific), 0.5 mM 3-isobutyl-1-methylxanthine (Enzo), 7.5 mM creatine phosphate (Sigma-Aldrich), 0.2 mg/mL creatine phosphokinase (Sigma-Aldrich), 1 mM sodium nitroprusside (Sigma-Aldrich), and 1 mM GTP (Sigma-Aldrich) at 37°C. After 10 minutes, the reaction was terminated using HCl (Sigma-Aldrich) to a final concentration of 0.1 N. Samples were dried using a Speed-Vac, and pellets were resuspended in 100–200 μL of cGMP EIA buffer (Cayman Chemical, Ann Arbor, MI). cGMP levels in the samples were measured in duplicate using a commercially available EIA kit (Cayman Chemical). Results were measured using a Bio-Rad iMark automated plate reader (Hercules, CA) at 405 nm [6 (link), 23 (link)]. sGC activity results are shown as pmol cGMP/minute/mg total protein.
Perez M., Lee K.J., Cardona H.J., Taylor J.M., Robbins M.E., Waypa G.B., Berkelhamer S.K, & Farrow K.N. (2017). Aberrant cGMP signaling persists during recovery in mice with oxygen-induced pulmonary hypertension. PLoS ONE, 12(8), e0180957.
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6

In Vitro Ubiquitination Assay of DCX

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In vitro ubiquitination assays were done as previously described (68 (link)), but modified for 48-well plates. Briefly, the ubiquitination reaction mixture contained 125 ng of E1 enzyme (Boston Biochem), 250 ng of E2 enzyme (Boston Biochem), immunopurified Cul3–KLHL15 E3 complex, 10 μg of Myc-ubiquitin (Boston Biochem), 1 μM ubiquitin aldehyde (Boston Biochem), 10 mM MgCl2, 2 mM DTT, 10 mM creatine phosphate (Sigma), 0.5 mg/ml creatine phosphokinase (Sigma), and 20 μg of purified proteins including DCX-HaloTag-His6 (WT or Y259L) or Halo-His6 as control protein. The ubiquitination reactions were diluted to a final volume of 0.25 ml in 50 mM HEPES, pH 7.5, initiated upon addition of 5 mM ATP (Thermo Scientific), and incubated for up to 2 h at 37 °C on a titer plate shaker (constant speed 3, Lab-Line Instruments, Inc). Thirty-microliter reaction mixtures were sampled at the indicated times and immediately terminated in 4× Laemmli buffer. Proteins were then resolved on 8% gels by SDS-PAGE, and ubiquitinated proteins were detected by immunoblotting with Myc-Tag antibody.
Relative DCX ubiquitination in vitro was quantified by dividing Myc-Tag signals by DCX signals and GFP (KLHL15) signals in the same lane and normalizing to the zero time point (set to 1). The enzymatic kinetics was plotted in Michaelis–Menten model using GraphPad Prism.
Song J., Merrill R.A., Usachev A.Y, & Strack S. (2020). The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis. The Journal of Biological Chemistry, 296, 100082.
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7

In Vitro Hepatitis B Virus cccDNA Repair

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60 ng of RrcDNA or NeutrAvidin-RrcDNA complex, or 1–5 ng (estimated by quantitative PCR) of deproteinated virion derived rcDNA were incubated with yeast cell extract in reaction buffer consisting of 20 mM Tris-HCl (pH 7.6), 50 mM KCl, 0.1 mM dNTP, 5 mM MgCl2, 1 mM reduced glutathione, 2.6 mM ATP, 26 mM phosphocreatine disodium (Sigma Aldrich, St. Louis, MO), and 6 μg/ml creatine phosphokinase (Sigma Aldrich, St. Louis, MO). The mixture was incubated at 30°C for 60 min before the reaction was stopped by addition of SDS and EDTA to final concentrations of 0.5% (w/vol) and 25 mM, respectively. The solution was subsequently treated with RNase A and proteinase K for 1 hour each. The solution was then extracted by phenol-chloroform (1:1, vol/vol) and repair product precipitated by ethanol and dissolved in TE buffer consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. To monitor cccDNA formation, the repair products were loaded onto a 0.7% (w/vol) agarose gel containing 0.05 μg/ml ethidium bromide and run at 4 V/cm for 2 hours before the gel was visualized on Typhoon™ FLA 9500 (GE Healthcare Lifesciences, Chicago, IL). The intensity of DNA bands was quantified by ImageJ.
For the repair reaction using deproteinated rcDNA (dp-rcDNA), cccDNA was detected by Southern blotting as describe in the Southern blotting section.
Wei L, & Ploss A. (2020). Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation. Nature microbiology, 5(5), 715-726.
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8

Purification and Characterization of E. coli ClpP and ClpX

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E. coli ClpP, single-chain E. coli ClpX variants, and ssrA-tagged GFP substrates were expressed as described22 (link),26 (link),33 (link). Briefly, proteins were purified by Ni+2-NTA (Qiagen) affinity chromatography, desalted into a low ionic strength buffer on a PD-10 column (GE Healthcare), purified further by ion-exchange chromatography, run on a HiLoad 16/60 Superdex 200 size-exclusion column (Amersham), and stored frozen at −80 °C in PD buffer (25 mM HEPES, pH 7.6, 100 mM KCl, 20 mM MgCl2, 10% glycerol (v/v)). Degradation assays were performed at 30 °C in PD buffer supplemented with 4 mM ATP and a regeneration system consisting of 16 mM creatine phosphate (MP Biomedicals) and 0.32 mg/mL creatine phosphokinase (Sigma). GFP degradation and extraction of the ssrA-tagged strand of split SFGFP-10/11-ssrA were quantified by loss of 511-nm fluorescence after excitation at 400 or 467 nm26 (link). Rates of ATP hydrolysis were determined using an NADH coupled assay in PD buffer at 30 °C34 (link). The binding of ClpX variants to ClpP was assayed by changes in the rate of cleavage of a decapeptide35 (link).
Iosefson O., Nager A.R., Baker T.A, & Sauer R.T. (2015). Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine. Nature chemical biology, 11(3), 201-206.
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9

Nitrogenase Activity Assay in Vitro

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Nitrogenase activity was assessed in vitro by measuring specific activities for H2 and NH3 formation under an N2 or Ar atmosphere. Working under an Ar atmosphere, varying amounts of AnfH2 were dissolved in an anaerobic solution of 50 mM Tris (pH 7.8), 10 mM sodium dithionite, 3.5 mM ATP, 7.87 mM MgCl2, 44.59 mM creatine phosphate and 0.20 mg ml−1 creatine phosphokinase (Sigma-Aldrich, C3755). The reaction vials were sealed by crimping them with butyl rubber stoppers, and the headspace was exchanged to N2 or Ar. Next, the reactions were initialized by adding 0.1 mg of Anf(DGK)2 up to a total volume of 700 µl and were allowed to proceed for 8 min at 30 °C with moderate shaking at 250 r.p.m. Reactions were quenched with 300 µl of 400 mM Na2EDTA solution (pH 8.0), and the amounts of formed H2 and NH3 were analyzed as described below.
Schmidt F.V., Schulz L., Zarzycki J., Prinz S., Oehlmann N.N., Erb T.J, & Rebelein J.G. (2023). Structural insights into the iron nitrogenase complex. Nature Structural & Molecular Biology, 31(1), 150-158.
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10

In vitro Ubiquitination Assay of GmPUB1

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In vitro ubiquitination assays were conducted as described previously (Yang et al., 2006 (link)) with slight modifications. 1 μg of purified wild type and mutant His-GmPUB1 proteins, 50 ng of human recombinant E1 enzyme (Boston Biochem, Cambridge, MA), 150 ng of E2 UbcH5b (Boston Biochem, Cambridge, MA), 2 μg of HA-Ub (Boston Biochem, Cambridge, MA) were incubated in a final volume of 30 μl reaction buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.2 mM DTT, 3 mM ATP, 10 mM creatine phosphate and 0.1 unit of creatine phosphokinase (Sigma-Aldrich, St. Louis, MO). Reactions were incubated at 30°C for 2 h and stopped by boiling with 4X SDS-PAGE loading buffer for 5 min. 15 μl of each reaction was analyzed by electrophoresis on 10% SDS-PAGE and subjected to immunoblot analysis. Ubiquitination was detected with anti-HA antibody (Invitrogen, Carlsbad, CA) and anti-His (Amersham Bioscience, PA) antibody.
Li S., Hanlon R., Wise H., Pal N., Brar H., Liao C., Gao H., Perez E., Zhou L., Tyler B.M, & Bhattacharyya M.K. (2021). Interaction of Phytophthora sojae Effector Avr1b With E3 Ubiquitin Ligase GmPUB1 Is Required for Recognition by Soybeans Carrying Phytophthora Resistance Rps1-b and Rps1-k Genes. Frontiers in Plant Science, 12, 725571.
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