Mouse/Rat/Porcine/Canine IGF-II/IGF2 Quantikine ELISA Kit Summary

Product Summary

The Quantikine® Mouse/Rat/Porcine/Canine IGF-II Immunoassay is a 3.5 hour solid-phase ELISA designed to measure IGF-II in serum-free cell culture supernates and serum. It contains E. coli-expressed recombinant IGF-II and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant factor. Results obtained using natural IGF-II showed linear curves that were parallel to the standard curves obtained using the Quantikine® kit standards. These results indicate that this kit can be used to determine relative mass values for natural IGF-II.

Preparation and Storage

Background: IGF-II/IGF2

Insulin-like growth factor (IGF)-I (also known as somatomedin C and somatomedin A) and IGF-II (multiplication stimulating activity or MSA) belong to the family of insulin-like growth factors that are structurally homologous to proinsulin. Mature IGF-I and IGF-II share approximately 70% sequence identity. Both IGF-I and IGF-II are expressed in many tissues and cell types and may have autocrine, paracrine and endocrine functions. Mature IGF-I and IGF-II are highly conserved between the human, bovine and porcine proteins (100% identity), and exhibit cross-species activity.

The IGFL (insulin-like growth factor-like) family includes four small (~11 kDa), probably secreted family members in humans and one in mouse. This family shares A and B chain cysteine motifs with the IGF superfamily, and has an additional cysteine motif within an uncleaved region corresponding to the C peptide of the IGF family.

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.