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Pyrophosphate assay kit

Manufactured by Abcam
Sourced in China, United States

The Pyrophosphate assay kit is a quantitative colorimetric assay designed to measure pyrophosphate (PPi) levels in various samples. The kit utilizes a coupled enzymatic reaction to produce a colored product, the absorbance of which is proportional to the PPi concentration in the sample.

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4 protocols using pyrophosphate assay kit

1

Purification and Characterization of Pn Protein

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The pn CDS was cloned into the pETDuet-1vector and expressed in the Rosetta™ 2(DE3) Singles™ Competent Cells (Merck Millipore) induced with 1 mM IPTG (Merck Millipore) at 16°C overnight. The Ni-NTA Fast Start kit (Qiagen) was used to purify the N-terminal His-tagged Pn protein. After dialysis (10 mM Tris–HCl, pH 8.0), the protein aliquot was saved for future use. Pyrophosphate activity and cyclic nucleotide phosphodiesterase activity were determined using a Pyrophosphate Assay Kit (Abcam) and a Cyclic Nucleotide Phosphodiesterase Assay kit (Enzo Life Sciences). A total of 20 mM NaF was used as the pyrophosphate inhibitor, and 40 μM IBMX was used as the PDE inhibitor.
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2

ADTP and AL Bioassay Protocols

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ADTP and AL were purchased from Solarbio Biotech Co. (Beijing, China). ADTP was prepared as 0.05 mg/mL and 0.1 mg/mL being the low and high concentration respectively. AL was prepared as 0.06 mg/mL and 0.12 mg/mL being the low and high concentration respectively. Pyrophosphate assay kit was purchased from Abcam Co. (Shanghai, China). The kits for lipid profiles, the ELISA kits for inflammatory factors and the ALP activity kit were purchased from Nanjing Jiancheng Bioengineering Co. (Nanjing, China).
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3

Biochemical Characterization of SUMO E1 Enzymes

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Biochemical activity of SUMO-specific E1 enzymes was examined by ATPase assay and in-vitro gel-shifting assays. ATP hydrolysis activity of E1 enzymes was measured by using pyrophosphate assay kit (Abcam Inc.). E1 (0.05 μM), 0.25 μM E2, 12.5 μM SUMO-1 and 1 mM ATP with N106 or DMSO were used for the assays. The E1-thioester formation was analysed in reaction mixtures containing 0.05 μM E1, 12.5 μM SUMO-1 and 1 mM ATP with N106 or DMSO. The E1-dependent E2-SUMO-1 thioester formation was assessed using reaction containing 0.05 μM E1, 0.25 μM E2, 12.5 μM SUMO-1 and 1 mM ATP with N106 or DMSO. Assays were incubated at 37 °C for 30 min and were mixed with non-reducing sample buffer, and analysed in non-reducing condition. Western blotting using antibodies against SAE2 and Ubc9 detected the SUMO conjugations. To determine the effect of N106 on Ubiquitin-activating enzyme E1 activity, similar assays were performed (Supplementary Fig.6). Inorganic pyrophosphate detection assays, Ubiquitin-E1 conjugation and Ubiquitin-E2 conjugation were performed as previously described32 (link). Human recombinant proteins (WT SAE1/SAE2, Ubc9, SUMO-1, Ubiquitin and UbcH5A) and reaction buffers were purchased from R&D Systems. N106 stock solution for in-vitro assays was prepared in DMSO (Sigma-Aldrich).
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4

Biochemical Characterization of SUMO E1 Enzymes

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Biochemical activity of SUMO specific E1 enzymes was examined by ATPase assay and in vitro gel-shifting assays. ATP hydrolysis activity of E1 enzymes was measured by using pyrophosphate assay kit (Abcam Inc., MA, USA). 0.05 µM E1, 0.25 µM E2, 12.5 µM SUMO-1, and 1 mM ATP with N106 or DMSO were used for the assays. The E1-thioester formation was analyzed in reaction mixtures containing 0.05 µM E1, 12.5 µM SUMO-1, and 1 mM ATP with N106 or DMSO. The E1 dependent E2-SUMO-1 thioester formation was assessed using reaction containing 0.05 µM E1, 0.25 µM E2, 12.5 µM SUMO-1, and 1 mM ATP with N106 or DMSO. Assays were incubated at 37°C for 30 minute and were mixed with non-reducing sample buffer and analyzed in non-reducing condition. Western blot using antibodies against SAE2 and Ubc9 detected the SUMO conjugations. To determine the effect of N106 on Ubiquitin-activating enzyme E1 activity, similar assays were performed (Supplementary Fig.6). Inorganic pyrophosphate detection assays, Ubiquitin-E1 conjugation, and Ubiquitin-E2 conjugation were performed as previously described32 (link). Human recombinant proteins (wild type SAE1/SAE2, Ubc9, SUMO-1, Ubiquitin, UbcH5A) and reaction buffers were purchased from R&D systems. N106 stock solution for in vitro assays was prepared in DMSO (Sigma-Aldrich, MO, USA).
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