Human IGF-II R/IGF2R DuoSet ELISA
Human IGF-II R/IGF2R DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human IGF-II R. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
DuoSet Ancillary Reagent Kit 3 (5 plates): (Catalog # DY009) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 3.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY004), or 5% Tween 20 in PBS, 7.2-7.4, 0.2 μm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Human IGF-II R ELISA Standard Curve
Preparation and Storage
Background: IGF-II R/IGF2R
IGF-I receptor is a disulfide-linked heterotetrameric transmembrane protein consisting of two alpha and two beta subunits. Both the alpha and beta subunits are encoded within a single receptor precursor cDNA. The proreceptor polypeptide is proteolytically cleaved and disulfide-linked to yield the mature heterotetrameric receptor. The alpha subunit of IGF-I receptor is extracellular while the beta subunit has an extracellular domain, a transmembrane domain and a cytoplasmic tyrosine kinase domain. The IGF-I receptor is highly expressed in all cell types and tissues.
IGF-II R is a type I transmembrane glycoprotein that contains a 2,264 amino acid (aa) extracellular region, a 23 aa transmembrane segment segment and a 124 aa cytoplasmic tail. IGF-II R regulates many diverse biological functions that range from intracellular trafficking to the internalization of extracellular factors and modulation of cellular responses. It delivers newly synthesized M6P-tagged lysosomal enzymes from the trans-golgi network to endosomes, and facilitates the clearance of extracellular lysosomal and matrix degrading enzymes by internalization into clathrin-coated vesicles and delivery into endosomes. With respect to IGF-II biology, It would appear that IGF-II R is principally a regulator of local IGF-II levels, targeting IGF-II for destruction in lysosomes.
The heterotetrameric receptors for insulin (INS R) and IGF-I (IGF-I R) are receptor tyrosine kinases that consist of two ligandbinding alpha subunits and two beta subunits. Ligand binding induces autophosphorylation on multiple tyrosine residues of beta subunits. Phosphorylation of Tyr1162 and 1163 on INS R and Tyr1135 and 1136 on IGF-I R stimulates intrinsic kinase activity.
GENERAL ELISA PROTOCOL
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block plates by adding 300 μL of Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
- Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Human IGF-II R/IGF2R DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 2
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Expression, purification, and characterization of human mannose-6-phosphate receptor - Extra cellular domain from a stable cell line utilizing a small molecule biomimetic of the mannose-6-phosphate moiety
Authors: B Dwyer, D Lundberg, A Iskenderia, B Strack-Log, B Pescatore, AW Norton, J Xu, M Meiyappan, MF Concino, B Zhang
Protein Expr. Purif., 2020-02-03;0(0):105589.
Sample Types: Cell Culture Supernates
Insulin-like growth factors and related proteins in plasma and cerebrospinal fluids of HIV-positive individuals.
Authors: Suh H, Lo Y, Choi N, Letendre S, Lee S
J Neuroinflammation, 2015-04-15;12(0):72.
Sample Types: Plasma
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