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96-Well Plate
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48-602MAG
Buffer Detection Kit for Magnetic Beads
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A specific and sensitive assay for measuring active human, mouse, or rat Insulysin (IDE) in cell lysates, tissue extracts, and biological fluids. IDE is a major enzyme responsible for degrading insulin hence critical for glucose, lipid, and protein metabolism, as well as for cell growth and differentiation. It also plays an important role in both diabetes and Alzheimer's disease.
Catalogue Number
CBA079
Brand Family
Calbiochem®
Application Data
Recombinant rat and human IDE activity was measured as outlined in the Detailed Protocol above using a 4-h incubation at 37°C. The data is presented as Relative Fluorescence Units. IDE Activity in Biological Samples. Cells (cell types are denoted in the figure) were cultured in DMEM or RPMI medium supplemented with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the manufacturer's recommended protocol. Human spleen extract was prepared by homogenizing tissue in 170 mM NaCl and 2 mM EDTA. Protein concentration was determined using a BCA protein assay. Activity was assessed as outlined in the Detailed Protocol above. Fluorescence intensity was measured at an excitation wavelength of 320 nm and an emission wavelength of 405 nm and expressed per mg total protein.
Materials Required but Not Delivered
• Distilled H2O • Pipettors or multi-channel pipettor carefully calibrated to the target volume • 37°C incubator • Fluorescent plate reader capable of measuring fluorescence at an excitation wavelength of 320-330 nm and an emission wavelength of 400-410 nm
References
References
Li, Q., et al. 2006. Cell127, 305. Shen, Y., et al. 2006. Nature443, 870. Song, E.S. and Hersh, L.B., 2005. J. Mol. Neurosci.25, 201. Farris, W., et al. 2003. PNAS USA100, 4162. Morelli, L., et al. 2003. J. Biol. Chem.278, 23221. Song, E.S., et al. 2001. J. Biol. Chem.276, 1152.
Product Information
Detection method
Fluorescence
Form
96 Tests
Format
96-well plate
Kit contains
Control Rat Recombinant IDE, Anti-Insulysin/IDE Coated 96-Well Plate, Substrate, Assay Buffer, 10X Sample Buffer, Plate Sealer, and a user protocol.
Positive control
Recombinant rat IDE
Biological Information
Assay range
50-1000 ng/ml
Assay time
3.5 h
Sample Type
Cell lysates, tissue extracts, biological fluids
Product Usage Statements
Intended use
The Calbiochem® InnoZyme™ Insulysin/IDE Immunocapture Activity Assay Kit is designed to measure human, mouse, or rat insulysin activity in biological samples such as cell lysates, tissue extracts, and biological fluids.
Storage and Shipping Information
Ship Code
Dry Ice Only
Toxicity
Regulatory Review
Storage
Multiple storage requirements
Storage Conditions
Upon arrival store the entire contents of the unopened kit at -20°C or -70°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
Table 1: Storage
Note: Following initial use the Control, Rat Recombinant IDE should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles.
Do not freeze
Ok to freeze
Supplemental Information
Kit contains
Control Rat Recombinant IDE, Anti-Insulysin/IDE Coated 96-Well Plate, Substrate, Assay Buffer, 10X Sample Buffer, Plate Sealer, and a user protocol.
Li, Q., et al. 2006. Cell127, 305. Shen, Y., et al. 2006. Nature443, 870. Song, E.S. and Hersh, L.B., 2005. J. Mol. Neurosci.25, 201. Farris, W., et al. 2003. PNAS USA100, 4162. Morelli, L., et al. 2003. J. Biol. Chem.278, 23221. Song, E.S., et al. 2001. J. Biol. Chem.276, 1152.
Upon arrival store the entire contents of the unopened kit at -20°C or -70°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
Table 1: Storage
Note: Following initial use the Control, Rat Recombinant IDE should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles.
Intended use
The Calbiochem® InnoZyme™ Insulysin/IDE Immunocapture Activity Assay Kit is designed to measure human, mouse, or rat insulysin activity in biological samples such as cell lysates, tissue extracts, and biological fluids.
Background
Insulysin or Insulin Degrading Enzyme (IDE), also known as Insulinase, is an evolutionarily conserved, ubiquitously expressed, 110 kDa zinc metalloprotease that plays an important role in both diabetes and Alzheimer's disease. The enzyme is primarily cytosolic, however a fraction of the enzyme is proximal. IDE is also located on the cell surface, but can also be secreted. IDE is the major enzyme responsible for degrading insulin and, thus, is critical for glucose, lipid, and protein metabolism, as well as for cell growth and differentiation. Other physiological substrates include glucagons, insulin-like growth factor I and II, amylin, atrial natriuretic peptide, β-endorphin, transforming growth factor-α, and amyloid-β (Aβ). IDE also degrades the Aβ genetic variants that result in amino acid substitutions that lead to pathogenesis. Homozygous deletion of the IDE gene in mice results in an increase in the level of brain Aβ by up to 65% and a 180% increase in serum insulin levels and impaired glucose tolerance, which are hallmarks of type 2 diabetes mellitus (DM2). While hyperinsulinemia and glucose intolerance are hallmarks of DM2, the role of IDE in this process is not well understood. In human genetic studies, the IDE region of chromosome 10q has been linked to late-onset AD and DM2. In a recent study IDE was identified as a cellular receptor for Varicella Zoster virus glycoprotein E (gE), which is the protein responsible for viral entry and cell-to-cell spread.
The activity of IDE toward a vast array of physiological substrates can be partially explained by the detailed crystal structure of the enzyme. The structural data revealed that IDE is shaped like a clam shell, consisting of two bowl-shaped halves connected by a flexible hinge, which allows the enzyme to exist in two conformations, closed and open. During catalytic processing of substrates, the enzyme switches from the open structure to the closed configuration and back to the open structure as IDE binds, catalyzes, and then releases its substrate, respectively. The extended hydrogen bonding between the two halves of IDE creates a "latch" that acts to maintain the enzyme in the closed state. Mutations that promote the open conformation have been shown to improve the protease's efficiency in cleaving the substrate by as much as 40-fold. The high-resolution structure of the IDE opening region and catalytic sites opens possibilities for development of functional activators, which may provide a structure-based approach to the pharmacological targeting of this protease in both Alzheimer's disease and type 2 diabetes.
Principles of the assay
The Calbiochem® InnoZyme™ Insulysin/IDE Immunocapture Activity Assay Kit uses an affinity purified polyclonal antibody that recognizes human, mouse, and rat insulysin, immobilized on a 96-well plate to capture the enzyme. Activity of captured insulysin is measured using a FRET substrate, Mca-GGFLRKHGQ-EDDnp. Cleavage of the scissile amide bond between R and K releases the fluorophore from the quenching molecule, Dnp, resulting in an increase in fluorescence. The increase in fluorescence is measured using an excitation wavelength of 320 nm and an emission wavelength of 405 nm.
Materials provided
• Anti-Insulysin/IDE Coated 96-Well Plate (Kit Component No. JA9374-1EA): 1 plate, 96-wells, supplied as six 2 x 8 well strips, coated with an antibody specific for human insulysin (also cross-reacts with mouse and rat insulysin) • Control, Rat Recombinant IDE (Kit Component No. JA9375-30UL): 1 vial, 30 µl supplied at 100 µg/ml • Substrate (Kit Component No. JA9376-120UL): 1 vial, 120 µl, supplied as 2 mM Mca-GGFLRKHGQ-EDDnp in DMSO • Assay Buffer (Kit Component No. JA9377-20ML): 1 bottle, 20 ml • 10X Sample Buffer (Kit Component No. JA9378-20ML): 2 bottles, 20 ml each, supplied as a 10X solution • Plate Sealer: 2 each
Materials Required but not provided
• Distilled H2O • Pipettors or multi-channel pipettor carefully calibrated to the target volume • 37°C incubator • Fluorescent plate reader capable of measuring fluorescence at an excitation wavelength of 320-330 nm and an emission wavelength of 400-410 nm
Preparation
• Samples: Dilute samples with Sample Buffer (1X) as needed.
Suggestion in sample preparation:
• The protein concentration in cell lysates should be ≥5 mg/ml
• The dilution factor for cell lysates (at ≥5 mg/ml) is approximately 1:5
Reagent preparation
Warm all reagents to room temperature (15-25°C) just prior to use.
• Sample Buffer (1X): Dilute 10X Sample Buffer 1:10 with distilled water. For example, to prepare 200 ml Sample Buffer (1X), add 20 ml 10X Sample Buffer to 180 ml distilled water.
• Diluted Control: Dilute the Control, Rat Recombinant IDE 100-400-fold with chilled Sample Buffer (1X). Maintain the Diluted Control on ice at all times.
Important: Following initial thaw the Control, Rat Recombinant IDE should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles.• Substrate (1X): Dilute the Substrate 1:100 with Assay Buffer. For example, to prepare enough Substrate (1X) for the entire 96-well plate, add 100 µl Substrate to 9.90 ml Assay Buffer.
Detailed protocol
It is recommended that all samples and standards be assayed in duplicate.
1. Remove the desired number of strips from the 96-Well Plate, return the remaining strips to the foil pouch, and close the re-sealable edge. 2. Wash the strips by pipetting 350 µl Sample Buffer (1X) into each well. Empty the contents into the sink. Repeat for a total of 2 washes. Following the final wash, tap the inverted plate gently on paper towels to remove excess Sample Buffer (1X). 3. Add 100 µl Diluted Control and samples to designated wells. Prepare Blank wells by adding 100 µl Sample Buffer (1X) to designated wells. 4. Cover with a Plate Sealer and incubate the plate for 60 min at room temperature with gentle shaking. 5. Wash the plate by adding 350-400 µl Sample Buffer (1X) to each well. Following the wash discard the contents of the wells by shaking the contents of the wells into the sink; invert the plate and tap on a paper towel to remove residual liquid. Repeat for a total of 5 washes 6. Add 100 µl Substrate (1X). Cover the plate with a Plate Sealer and incubate for 2-4 h at 37°C. Protect from light. 7. Read the fluorescence at an excitation wavelength of 320 nm and an emission wavelength of 405 nm.
Calculations
Correct the fluorescence value of all Diluted Controls and samples by subtracting the value of the blank from each reading. Calculate the mean fluorescence value for each duplicate Diluted Control and sample. Plot the data as Relative Fluorescence Units (RFU) as show in the figure below.
Assay characteristics and examples
Figure 1: Activity of recombinant rat and human IDE.
Recombinant rat and human IDE activity was measured as outlined in the Detailed Protocol above using a 4-h incubation at 37°C. The data is presented as Relative Fluorescence Units.
Figure 2: IDE Activity in Cell Lysates and Tissue Extracts
IDE Activity in Biological Samples. Cells (cell types are denoted in the figure) were cultured in DMEM or RPMI medium supplemented with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the manufacturer's recommended protocol. Human spleen extract was prepared by homogenizing tissue in 170 mM NaCl and 2 mM EDTA. Protein concentration was determined using a BCA protein assay. Activity was assessed as outlined in the Detailed Protocol above. Fluorescence intensity was measured at an excitation wavelength of 320 nm and an emission wavelength of 405 nm and expressed per mg total protein.
Table 2: Cross-Reactivity of InnoZyme™ Insulysin/IDE Immunocapture Activity Assay K
The activity of recombinant human and rat neprilysin (31-1000 ng/ml) was assessed as outlined in the Detailed Protocol above. No cross-reactivity was observed with neprilysin.
Assay Range
50-1000 ng/ml
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. InnoZyme™, CytoBuster™, and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.
