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CBA085
Sigma-AldrichInnoZyme™ DPP IV Immunocapture Activity Assay
Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase. The natural substrate of this enzyme include chemokines, cytokines, endomorphines, hormones and the pancreatic polypeptide of the PACAP/glucagons peptide family. Of particular interest is the ability of DPP IV to regulate incretin hormones. Since these hormones are deactivated by DPP IV, a multivalent method of treatment for type 2 diabetes and, more generally, in glucose metabolism disorders, is opened by the inhibition of this protease.
Catalogue Number
CBA085
Brand Family
Calbiochem®
Application Data
The activities of recombinant human DPP II, FAP, and DPP I were assessed as outlined in the Detailed Protocol above. Human recombinant DPP IV activity was measured as outlined in the Detailed Protocol above using a 1-h incubation at 37°C. The data is presented as Relative Fluorescence Units. Cells (cell types are denoted in the figure) were cultured in DMEM 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 with 170 mM NaCl, 2 mM EDTA. Human plasma (pooled samples) was obtained from commercial sources. The concentration of active DPP IV was determined by extrapolating the RFU values to the standard curve prepared with human recombinant DPP IV. Protein concentration in cell lysates and tissue extract 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 360 nm and an emission wavelength of 460 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 360 nm and an emission wavelength of 460 nm
References
References
McIntosh, C.H. 2008. Front. Biosci. 13, 1753. Thompson, M.A., et al. 2007. Mini Rev Med Chem.7, 253. Demuth, H.U., et al. 2005. Biochim Biophys Acta.1751, 33. Gallwitz, B. 2005. Rev Diabetic Stud.2, 61. Pro, B., and Dang, N.H. 2004. Histol Histopathol.19, 1345. Durinx, C., et al. 2000. Eur J Biochem.267, 5608.
Product Information
Detection method
Fluorescence
Form
96 Tests
Format
96-well plate
Kit contains
Anti-DPP IV Antibody 96-well Coated Plate, DPP IV Control, DPP IV Substrate, Assay Buffer, 10X Sample Buffer, Plate Sealer, and a user protocol
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable gloves and eye/face protection. Dispose of this material and its container at hazardous or special waste collection point.
Product Usage Statements
Intended use
The Calbiochem® InnoZyme™ DPP IV Immunocapture Activity Assay is intended for the measurement of human DPP IV activity in biological samples such as cell lysates, tissue extracts, and biological fluids.
Storage and Shipping Information
Ship Code
Dry Ice Only
Toxicity
Irritant
Storage
-20°C
Storage Conditions
Upon arrival store the unopened kit at -20°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
Table 1: Storage Conditions
Note: Following initial use the Control should be dispensed into aliquots and stored at -20°C or -70°C. Avoid freeze/thaw cycles.
Do not freeze
Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains
Anti-DPP IV Antibody 96-well Coated Plate, DPP IV Control, DPP IV Substrate, Assay Buffer, 10X Sample Buffer, Plate Sealer, and a user protocol
Specifications
Global Trade Item Number
Bestellnummer
GTIN
CBA085
0
Documentation
InnoZyme™ DPP IV Immunocapture Activity Assay Analysenzertifikate
Titel
Chargennummer
CBA085
Literatur
Übersicht
McIntosh, C.H. 2008. Front. Biosci. 13, 1753. Thompson, M.A., et al. 2007. Mini Rev Med Chem.7, 253. Demuth, H.U., et al. 2005. Biochim Biophys Acta.1751, 33. Gallwitz, B. 2005. Rev Diabetic Stud.2, 61. Pro, B., and Dang, N.H. 2004. Histol Histopathol.19, 1345. Durinx, C., et al. 2000. Eur J Biochem.267, 5608.
Anwenderprotokoll
Revision
26-June-2012 JSW
Form
96 Tests
Format
96-well plate
Detection method
Fluorescence
Species
human
Storage
Upon arrival store the unopened kit at -20°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
Table 1: Storage Conditions
Note: Following initial use the Control should be dispensed into aliquots and stored at -20°C or -70°C. Avoid freeze/thaw cycles.
Intended use
The Calbiochem® InnoZyme™ DPP IV Immunocapture Activity Assay is intended for the measurement of human DPP IV activity in biological samples such as cell lysates, tissue extracts, and biological fluids.
Background
Dipeptidyl peptidase IV (DPP IV), also known as CD26, gp110 DPP 4, or adenosine deaminase-binding protein (ADAbp), is a serine exopeptidase. Together with fibroblast activation protein α (FAPα), it belongs to the propyl oligopeptidase family, S9. DPP IV is structurally unrelated to dipeptidyl peptidase II (DPP II) and atractin, but possesses similar substrate specificity.
DPP IV is a membrane-bound, homodimeric, type II protein with a molecular weight of 110-150 kDa per subunit. The enzyme is bound to the membrane by a 22-amino acid transmembrane sequence. In addition to its role as a protease, the DPP IV has a long extracellular domain (738 residues) that exhibits receptor properties and binds to the extracellular matrix (ECM) via collagen or fibronectin. In humans, the DPP IV activity is ubiquitous in almost all organs and tissues, with the highest expression in kidney, small intestine and placenta; moderate expression in lung, spleen, and liver; and a low levels of expression in heart, pancreas, and brain. A soluble form of DPP IV that can be found in human serum has been characterized as a proteolytic derivative of the membrane-bound form of DPP IV.
DPPIV exhibits specificity toward a vast array of physiological substrates including chemokines, cytokines, endomorphins, hormones, and the pancreatic polypeptide of the PACAP/glucagons peptide family. This broad specificity suggests a role for DPPIV in many vital physiological processes that regulate pain, the immune system, the cardiovascular system, and metabolism. Recently DPPIV was the subject of intense research in diabetes due to its role in regulating incretin hormones, GLP-1 and GIP, which have a pleiotropic effect on glucose metabolism. Both hormones are deactivated simultaneously by DPP IV, enabling a multivalent method of treatment for diabetes and glucose metabolism disorders via the inhibition of this protease. In addition, DPP IV is not regulated by limited proteolysis or endogenous inhibitors, therefore specific DPP IV inhibitors should possess a strong therapeutic potential in various pathological processes.
Principles of the assay
The Calbiochem® InnoZyme™ DPP IV Immunocapture Activity Assay uses an affinity purified polyclonal antibody specific for human DPP IV, immobilized on a 96-well plate to capture the enzyme. Activity of captured DPP IV is measured using a fluorogenic substrate, Gly-Pro AMC. Cleavage of the peptide bond releases the free AMC group, resulting in an increase in fluorescence. The increase in fluorescence is measured using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
The assay is designed to specifically measured DPP IV activity in a variety of biological samples. No cross-reactivity was detected with DPP II, FAPα, or DPP I.
Materials provided
The kit provides all components required to perform the assay.
• Anti-DPP IV Antibody Coated 96-Well Plate (Kit Component No. JA9435-1EA): 1 plate, 96-wells, supplied as six 2 x 8 well strips, coated with an antibody specific for human DPP IV • Control, Human Recombinant DPP IV (Kit Component No. JA9436-1EA): 1 vial, 30 µl supplied at 1 µg/ml • Substrate (Kit Component No. JA9437-120UL): 1 vial, 120 µl, supplied as 20 mM Gly-Pro AMC in DMSO • Assay Buffer (Kit Component No. JA9438-20ML): 1 bottle, 20 ml • 10X Sample Buffer (Kit Component No. JA9378-20ML): 2 bottles, 20 ml each, supplied as 10X • 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 360 nm and an emission wavelength of 460 nm
Preparation
• Samples: Dilute samples with 1X Sample Buffer as needed.
Suggestion for 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 1:50 to 1:100.
• The dilution factor for human plasma is approximately 1:50-1:100.
Reagent preparation
Warm all reagents to room temperature (15-20°C) just prior to use.
• 1X Sample Buffer: Dilute 10X Sample Buffer 1:10 with distilled water. For example, to prepare 200 ml 1X Sample Buffer, add 20 ml 10X Sample Buffer to 180 ml distilled water.
• Diluted Control: Dilute the Control, Human Recombinant DPP IV 50-100-fold with chilled 1X Sample Buffer. Maintain the Diluted Control on ice at all times.
Important: Following initial thaw, the Control, Human Recombinant DPP IV should be dispensed into aliquots and stored at -20°C. Avoid freeze/thaw cycles.• 1X Substrate: Dilute the Substrate 1:100 with Assay Buffer. For example, to prepare enough 1X Substrate 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 resealable edge. 2. Wash the strips by pipetting 350 µl 1X Sample Buffer to the wells. 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 1X Sample Buffer. 3. Add 100 µl Diluted Control and samples to designated wells. Prepare Blank wells by adding 100 µl 1X Sample Buffer to designated wells. 4. Cover the plate with a Plate Sealer and incubate for 60 min at room temperature with gentle shaking. 5. Wash the strips by pipetting 350 µl 1X Sample Buffer to the wells. Empty the contents into the sink. Repeat for a total of 4-5 washes. Following the final wash, tap the inverted plate gently on paper towels to remove excess 1X Sample Buffer. 6. Add 100 µl 1X Substrate. Cover the plate with a Plate Sealer and incubate for 1-2 h at 37°C. Protect from light. 7. Read the fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 460 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 1 below.
Assay characteristics and examples
Figure 1: Activity of Human Recombinant DPP IV
Human recombinant DPP IV activity was measured as outlined in the Detailed Protocol above using a 1-h incubation at 37°C. The data is presented as Relative Fluorescence Units.
Figure 2: Active DPP IV in Biological Samples
Cells (cell types are denoted in the figure) were cultured in DMEM 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 with 170 mM NaCl, 2 mM EDTA. Human plasma (pooled samples) was obtained from commercial sources. The concentration of active DPP IV was determined by extrapolating the RFU values to the standard curve prepared with human recombinant DPP IV. Protein concentration in cell lysates and tissue extract 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 360 nm and an emission wavelength of 460 nm and expressed per mg total protein.
Assay Range
0.31-20 ng/ml
Assay Range Notes
The assay was standarized against recombinat, human DPP IV expressed in S. frugiperda insect cells.
Specificity
Table 2: Cross-Reactivity
The activities of recombinant human DPP II, FAP, and DPP I were assessed as outlined in the Detailed Protocol above.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. InnoZyme™ and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.
