Human IGF-I R/IGF1R DuoSet ELISA Summary

Product Summary

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human IGF-I 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.

Preparation and Storage

Background: IGF-I R/IGF1R

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.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.    Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.    Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.    Add 50 μL of Assay Diluent to each well.

4.    Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.    Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.    Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.    Aspirate and wash 5 times.

8.    Add 100 μL Substrate Solution to each well.

9.    Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.