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Creatine phosphate

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Creatine phosphate is a high-energy phosphate compound found in muscle cells. It serves as a readily available source of phosphate for the regeneration of adenosine triphosphate (ATP), the primary energy currency of the cell.

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Lab products found in correlation

Creatine phosphokinase, by Merck Group (4 mentions) Ni2 nta, by Qiagen (2 mentions) Pd 10 column, by GE Healthcare (2 mentions) Hiload 16 60 superdex 200 size exclusion column, by Cytiva (2 mentions) Spark plate reader, by Tecan (1 mentions) Adenosine diphosphate (adp), by Merck Group (1 mentions) Adenosine triphosphate (atp), by MP Biomedicals (1 mentions) Spectramax m5, by Molecular Devices (1 mentions) Adenosine triphosphate (atp), by Merck Group (1 mentions) Z ggl amc, by Enzo Life Sciences (1 mentions)

4 protocols using creatine phosphate

1

Purification and Characterization of E. coli ClpP and ClpX

Cited 2 times
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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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2

Purification and Characterization of E. coli ClpP and ClpX

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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3

In vitro Proteolysis Assay of GFP Substrates

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In vitro proteolysis assays were carried out at 30 °C in 384-well black NBS low-binding plates (Corning), using 0.25 μM EcoClpX6, 0.50 μM EcoClpP14, 2.5 mM ATP, and a regeneration system comprising 16 mM creatine phosphate (MP Biomedicals) and 0.32 mg/mL creatine phosphokinase (Sigma), in PD buffer, in a total reaction volume of 40 μL. Degradation of each GFP substrate was measured by monitoring loss of 511 nm emission following 488 nm excitation in a Tecan Spark plate reader. Initial velocities of enzymatic degradation were fit to a Michaelis-Menten equation in Prism (GraphPad).
Beardslee P.C, & Schmitz K.R. (2024). Toxin-based screening of C-terminal tags in Escherichia coli reveals the exceptional potency of ssrA-like degrons. bioRxiv.
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4

Biochemical Assays for ATPase and Protease Activity

Cited 1 time
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Assays were performed at 30°C in PD buffer (25 mM HEPES, 100 mM KCl, 10% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, pH 7.5) on a SpectraMax M5 microplate reader (Molecular Devices). ATPase assays included 2.5 mM ATP (unless otherwise indicated; Sigma) and a NADH-coupled regeneration system (Nørby, 1988 (link)). Protease assays included 2.5 mM ATP and a regeneration system comprising 16 mM creatine phosphate (MP Biomedicals) and 0.32 mg/mL creatine phosphokinase (Sigma). ATP hydrolysis was monitored via decrease in 340 nm NADH absorbance. Peptidase assays followed hydrolysis of a fluorogenic decapeptide, Abz-KASPVSLGYNO2D (Lee et al., 2010b (link)), by increase in 420 nm fluorescence upon 320 nm excitation, or of a fluorogenic tripeptide (Z-GGL-AMC; Enzo Life Sciences) by increase in 460 nm fluorescence upon 380 nm excitation. GFP-ssrA degradation was monitored by loss of 511 nm emission following excitation at 450 nm. Z-Ile-Leu and ADP were obtained from Sigma. ATPγS was obtained from EMD.
Schmitz K.R, & Sauer R.T. (2014). Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase. Molecular microbiology, 93(4), 617-628.
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