Human IGF2R ELISA Kit (HUEB1839)- High Sensitivity

Product Name:	Human Cation-independent mannose-6-phosphate receptor (IGF2R) ELISA Kit
SKU:	HUEB1839
Size:	96T
Target:	Human Cation-independent mannose-6-phosphate receptor (IGF2R)
Synonyms:	300 kDa mannose 6-phosphate receptor, Insulin-like growth factor 2 receptor, Insulin-like growth factor II receptor, M6P/IGF2 receptor, MPR 300, IGF-II receptor, M6P/IGF2R, CD222, CI Man-6-P receptor, MPRI
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/ml
Sensitivity:	0.174ng/mL

Intra CV:

7.8%

Inter CV:

11.6%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	107-119%	114-124%	101-110%	88-100%
EDTA Plasma(N=5)	97-106%	105-114%	101-110%	88-97%
Heparin Plasma(N=5)	86-97%	105-115%	113-122%	102-111%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	104	98-110
Plasma	106	100-112

Function:

Transport of phosphorylated lysosomal enzymes from the Golgi complex and the cell surface to lysosomes. Lysosomal enzymes bearing phosphomannosyl residues bind specifically to mannose-6-phosphate receptors in the Golgi apparatus and the resulting receptor-ligand complex is transported to an acidic prelyosomal compartment where the low pH mediates the dissociation of the complex. This receptor also binds IGF2. Acts as a positive regulator of T-cell coactivation, by binding DPP4.

Uniprot:	P11717
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Cation-independent mannose-6-phosphate receptor
Sub Unit:	Binds HA-I and HA-II plasma membrane adapters (By similarity). Interacts with DPP4; the interaction is direct. Binds GGA1, GGA2 and GGA3.
Research Area:	Immunology
Subcellular Location:	Lysosome membrane Single-pass type I membrane protein Colocalized with DPP4 in internalized cytoplasmic vesicles adjacent to the cell surface.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	IGF2R: a multifunctional type I transmembrane protein receptor that binds insulin-like growth factor 2 at the cell surface and mannose-6-phosphate-tagged proteins in the trans-Golgi network. Also known as the cation-independent mannose-6 phosphate (M6P) receptor. Clears IGF2 from the cell surface to attenuate signaling, and transports M6P-tagged lysosomal enzymes from the Golgi complex and the cell surface to lysosomes. M6P-tagged lysosomal enzymes bind specifically to mannose-6- phosphate receptors in the Golgi apparatus and the resulting receptor-ligand complex is transported to an acidic prelyosomal compartment where the low pH mediates the dissociation of the complex. Acts as a positive regulator of T-cell coactivation, by binding DPP4. Belongs to the MRL1/IGF2R family.
UniProt Protein Details:	Protein type:Membrane protein, integral; Receptor, misc.; Vesicle Chromosomal Location of Human Ortholog: 6q26 Cellular Component: cell surface; clathrin coat; early endosome; endocytic vesicle; endosome; extracellular space; focal adhesion; integral to plasma membrane; late endosome; lysosomal membrane; membrane; nuclear envelope lumen; perinuclear region of cytoplasm; trans-Golgi network; trans-Golgi network transport vesicle Molecular Function:enzyme binding; G-protein alpha-subunit binding; G-protein coupled receptor activity; glycoprotein binding; identical protein binding; insulin-like growth factor II binding; insulin-like growth factor receptor activity; mannose binding; phosphoprotein binding; protein binding; receptor activity; retinoic acid binding; transporter activity Biological Process: G-protein coupled receptor protein signaling pathway; insulin-like growth factor receptor signaling pathway; liver development; organ regeneration; positive regulation of apoptosis; post-embryonic development; receptor-mediated endocytosis; response to retinoic acid; signal transduction; spermatogenesis Disease: Hepatocellular Carcinoma
NCBI Summary:	This gene encodes a receptor for both insulin-like growth factor 2 and mannose 6-phosphate. The binding sites for each ligand are located on different segments of the protein. This receptor has various functions, including in the intracellular trafficking of lysosomal enzymes, the activation of transforming growth factor beta, and the degradation of insulin-like growth factor 2. Mutation or loss of heterozygosity of this gene has been association with risk of hepatocellular carcinoma. The orthologous mouse gene is imprinted and shows exclusive expression from the maternal allele; however, imprinting of the human gene may be polymorphic, as only a minority of individuals showed biased expression from the maternal allele (PMID:8267611). [provided by RefSeq, Nov 2015]
UniProt Code:	P11717
NCBI GenInfo Identifier:	317373416
NCBI Gene ID:	3482
NCBI Accession:	P11717.3
UniProt Secondary Accession:	P11717,Q7Z7G9, Q96PT5,
UniProt Related Accession:	P11717
Molecular Weight:	274,375 Da
NCBI Full Name:	Cation-independent mannose-6-phosphate receptor
NCBI Synonym Full Names:	insulin like growth factor 2 receptor
NCBI Official Symbol:	IGF2R
NCBI Official Synonym Symbols:	MPR1; MPRI; CD222; CIMPR; M6P-R; MPR300; CI-M6PR; MPR 300; M6P/IGF2R
NCBI Protein Information:	cation-independent mannose-6-phosphate receptor
UniProt Protein Name:	Cation-independent mannose-6-phosphate receptor
UniProt Synonym Protein Names:	300 kDa mannose 6-phosphate receptor; MPR 300; Insulin-like growth factor 2 receptor; Insulin-like growth factor II receptor; IGF-II receptor; M6P/IGF2 receptor; M6P/IGF2R; CD_antigen: CD222
Protein Family:	Cation-independent mannose-6-phosphate receptor
UniProt Gene Name:	IGF2R
UniProt Entry Name:	MPRI_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

ELISA	Antibodies
Human IGF2R / M6P ELISA Kit	Anti-IGF2R Antibody (CAB13022)
Human IGF2R (Insulin Like Growth Factor 2 Receptor) CLIA Kit (HUES00317)	Anti-M6PR Antibody (CAB3762)
Human IGF2R (Insulin Like Growth Factor 2 Receptor) ELISA Kit (HUES01652)	IGF2R Antibody (PACO01972)
	Phospho-IGF2R (S2409) Antibody (PACO04127)
	IGF2R Antibody (PACO04128)