TPS enzyme assays was conducted following a procedure previously described [36 (link)]. Full-length open reading frames of three CfTPS genes were synthesized (Genescript, Nanjing, China) and subcloned into the vector pET32a with an N-terminal his-tag (http://www.emdmillipore.com ). To express CfTPS proteins, each protein expression construct was transformed into the E. coli strain BL21 (DE3) CodonPlus (http://www.agilent.com ). An empty pET32a vector without any insert was used as a negative control. After inducing expression of recombinant proteins by isopropyl b-d -1-thiogalactopyranoside (IPTG) for 16 h at 18 °C, the cells were collected by centrifugation and disrupted by 6 × 30 s treatment with an XL2000 probe sonicator (output power 100 W, Misonix, Farmingdale, NY, USA) in ice-chilled extraction buffer. The catalytic activity of E. coli expressed recombinant CfTPS was performed by using the substrates geranyl diphosphate and (E,E)-farnesyl diphosphate. Terpene products were collected by SPME and analyzed by GC-MS. The assays were also performed with crude proteins extracted from E. coli expressing the negative control vector, but no terpene products were detected.
Xl2000 probe sonicator
Manufactured by Bioventus
Sourced in United States
The XL2000 probe sonicator is a laboratory equipment designed for the process of sonication. It generates high-frequency sound waves that are used to disrupt biological samples, such as cells or tissues, for various research and analytical applications.
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Lab products found in correlation
2 protocols using xl2000 probe sonicator
1
Recombinant CfTPS Enzyme Assays
2
Quantifying Itaconate Levels in Murine and Human Samples
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Itaconate levels in the mouse retina/vitreous and cultured murine BMDMs were measured by LC-MS/MS methods at the Pharmacology and Metabolomics Core, Karmanos Cancer Institute, Wayne State University. Briefly, cell pellets were re-suspended in 1 mL of 80% MeOH (cooled in an ice bath), sonicated with a Misonix XL-2000 probe sonicator, and centrifuged at 28,672 x g for 10min at 4°C. The supernatant was transferred into a new 2 mL centrifuge tube. The pellet was washed with 200 μL of 80% MeOH (ice bath) by vortexing thoroughly and centrifuged at 28,672 x g for 10min at 4°C. Supernatant from two rounds of extraction was combined and dried in a CentriVap Refrigerated Centrifugal Concentrator. The residue was reconstituted with 50 μL of ddH2O, and a series of 20-time dilution samples were prepared for paralleled quantification by Waters Acquity H-class UPLC system connected to a Xevo TQ-xS triple LC-MS/MS system. The itaconate levels in the patient vitreous were measured by LC-MS/MS as indicated above at the Indian Institute of Chemical Technology, Hyderabad, India.
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