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Butyrylcholinesterase Fluorescent Activity Kit (Invitrogen™)

The Butyrylcholinesterase Fluorescent Activity research-use-only kit is a fluorescent activity assay designed for the quantification and detection of butyrylcholinesterase activity in serum and plasma samples.

This complete, ready-to-use kit includes black 96-well plate(s), butyrylcholinesterase standard (200 mU/mL), butyrylcholinesterase substrate, and other components to perform the assay. A fluorescence 96-well microplate reader capable of reading fluorescent emission at 510 nm, with excitation at 390 nm, is required for use of this kit.

Performance characteristics
• Assay type: fluorescent activity kit
• Sample types: serum and plasma samples
• Sensitivity: 0.018 mU/mL
• Standard curve range: 0.3 mU/mL–20 mU/mL
• Reactivity: species independent

Background
Butyrylcholinesterase (BChE) belongs to the same structural class of proteins as acetylcholinesterase (AChE). The 440 kDa tetrameric glycoprotein is predominantly found in blood, kidneys, intestine, liver, lung, heart, and the central nervous system. Many species, such as human, horse, and mice exhibit high BChE activity in plasma, whereas rats have higher acetylcholinesterase activity in plasma1. BChE preferentially acts on butyrylcholine, but also hydrolyzes acetylcholine. This assay has been validated for serum, EDTA and heparin plasmas from a variety of species.
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Assay principle
The Butyrylcholinesterase Activity kit is designed to quantitatively measure butyrylcholinesterase (BChE) activity in a variety of samples. A human BChE standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. The kit utilizes a proprietary non-fluorescent detection reagent that covalently binds to the thiol product of the reaction between the BChE Substrate and BChE in the standards or samples, yielding a fluorescent product read at 510 nm in a fluorescent plate reader with excitation at 390 nm. The kit is suitable for measuring BChE activity in appropriately diluted serum or plasma from a number of species. It will also measure BChE in extracted tissue samples, CSF, and cell lysates.

BChE unit definition
One unit will hydrolyze 1.0 μmol of butyrylcholine to choline and butyrate per minute at pH 8.0 and 37˚C.

Related links
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EnzChek™ Myeloperoxidase (MPO) Activity Assay Kit (Invitrogen™)

Myeloperoxidase (MPO) is a unique peroxidase that, in addition to its peroxidation activity, also catalyzes the conversion of hydrogen peroxide (H2O2) and chloride (Cl-) to hypochlorous acid (HOCl). The EnzChek® Myeloperoxidase (MPO) Activity Assay Kit provides assays for the determination of both chlorination and peroxidation activities of MPO in solution and in cell lysates. For detection of chlorination, the kit includes nonfluorescent 3'-(p-aminophenyl) fluorescein (APF), which is selectively cleaved by hypochlorite (-OCl) to yield fluorescein. Peroxidation is detected using nonfluorescent Amplex® UltraRed reagent (A36006), which is oxidized by the H2O2-generated redox intermediates MPO-I and MPO-II to form a fluorescent product. The EnzChek® Myeloperoxidase Activity Assay Kit can be used to continuously detect these activities at room temperature over a broad dynamic range (1.5 to 200 ng/mL). The speed (30 minutes), sensitivity, and mix-and-read convenience make this kit ideal for measuring MPO activities and for high-throughput screening for MPO-specific inhibitors.

Active Cdc42 Pull-Down and Detection Kit (Thermo Scientific™)

The Thermo Scientific Active Cdc42 Pull-Down and Detection Kit is a complete kit for selective enrichment and detection of GTP-bound Cdc42 GTPase through specific protein interaction with the Pak1 protein-binding domain.

The Active Cdc42 Pull-Down and Detection Kit includes purified GST-Pak1 protein-binding domain (PBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Cdc42 primary antibody, sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH 3T3 cells, a cell line that is known to have robust Cdc42 activity.

Features of the Active Cdc42 Pull-Down and Detection Kit:

Highly sensitive and accurate—optimized reagents, specific anti-Cdc42 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
Validated—functionally tested for Cdc42 detection to ensure quality and performance
Compatible—effective with a variety of cell types from mouse, rat and human sources

Applications:
• Follow activation of Cdc42 GTPase during cell differentiation, migration, division, and cytoskeletal rearrangement
• Study the activation of Cdc42 during filopodia formation
• Monitor Cdc42 activity after stimulation with growth factors
• Screen small molecule inhibitors for their effects on Cdc42 activity

The Active Cdc42 Pull-Down and Detection Kit was validated for the function and specificity of the active Cdc42 enrichment method using cell lysates treated with GTPγS to activate endogenous Cdc42 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Cdc42 in the GTP-bound, active form, resulting in a strong signal when endogenous Cdc42 is present. GDP treatment pushes Cdc42 into the GDP-bound, inactive state, resulting in minimal or no signal regardless of Cdc42 protein levels. This kit is optimized for Western blot detection with an HRP-conjugated secondary antibody (Goat Anti-mouse IgG, Part No. 31430) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.

Cdc42 Background:
Rho family GTPases serve many cellular functions, including cell signaling, transcriptional regulation and organization of the actin cytoskeleton. This family of GTPases comprise Rho (RhoA, RhoB, and RhoC), Rac (Rac1, Rac2, Rac3, and RhoG), Cdc42 (Cdc42 and G25K), Rnd (Rnd1, Rnd2, and RhoE/Rnd3), RhoBTB family and the Miro family. These GTPases enable signal transduction from the plasma membrane to the cytosol through GPCR, tyrosine kinase, cytokine and adhesion receptors. Attachment to the plasma membrane is accomplished through geranylgeranyl lipid modifications at the carboxy-terminus of the protein. Cdc42 activation results in the polymerization of actin filaments and filopodia formation.

The Cdc42 subfamily of RhoGTPase has been less well-characterized than the Rho and Rac families. Both Cdc42 and Rac1 are activated through tyrosine receptor kinase signaling leading to SAPK and p38 stress kinase pathway activation. Cdc42 is also activated by the chemoattractant fMLP in neutrophils. Fibronectin activates Cdc42 and Rac1 to induce cell spreading, and stimulation with TNF-alpha and IL-1 results in changes in the actin cytoskeleton. There is significant cross-talk between Cdc42 and Rac1, as they act in overlapping pathways, and in some cases, Cdc42 may act upstream of Rac1 during signal transduction. Some of the main effector proteins of Cdc42 are Pak1, N-WASP and IQGAP. Pak1 is a kinase involved in the activation of JNK in the SAPK stress pathway. N-WASP is an effector that induces filopodia formation, and IQGAP interacts with F-actin filaments. In differentiating neurons, Cdc42 plays an active role in neurite outgrowth. However, the Rho family of GTPases can work agonistically during cell signaling and antagonistically during differentiation.

More Product Data
Measure activation of small GTPases via their specific downstream effectors