Mouse GP4/CD36 ELISA Kit (MOFI00859)- High Sensitivity

Product Name:	Mouse GP4 / CD36 ELISA Kit
Product Code:	MOFI00859
Size:	96 Assays
Alias:	CD36, Collagen R, FAT, GPIIIb, GPIV, SCARB3, SR-B3, Thrombospondin R, CD36 antigen, CD36 molecule, thrombospondin receptor, FATCHDS7, Fatty acid translocase, Glycoprotein IIIb, GP3Bthrombospondin receptor, GPIIIB, Leukocyte differentiation antigen CD36, PAS IV, PAS-4, Platelet collagen receptor, platelet glycoprotein 4, Platelet glycoprotein IV, scavenger receptor class B, member 3, Thrombospondin receptor
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse GP4/CD36 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse GP4/CD36 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse GP4/CD36 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-99	96
EDTA plasma(n=5)	87-104	94
UFH plasma(n=5)	86-99	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse GP4/CD36 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	88-103%	87-103%	88-104%
EDTA plasma(n=5)	82-101%	83-96%	82-99%
UFH plasma(n=5)	82-92%	84-100%	81-100%

Intra Assay:

CV <8%

Inter Assay:

CV <10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

Q08857

UniProt Protein Function:	CD36: Seems to have numerous potential physiological functions. Binds to collagen, thrombospondin, anionic phospholipids and oxidized LDL. May function as a cell adhesion molecule. Directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes. Binds long chain fatty acids and may function in the transport and/or as a regulator of fatty acid transport. Receptor for thombospondins, THBS1 AND THBS2, mediating their antiangiogenic effects. Defects in CD36 are the cause of platelet glycoprotein IV deficiency (PG4D)[MIM:608404]; also known as CD36 deficiency. Platelet glycoprotein IV deficiency can be divided into 2 subgroups. The type I phenotype is characterized by platelets and monocytes/macrophages exhibiting complete CD36 deficiency. The type II phenotype lacks the surface expression of CD36 in platelets, but expression in monocytes/macrophages is near normal. Genetic variations in CD36 are associated with susceptibility to coronary heart disease type 7 (CHDS7). Belongs to the CD36 family.
UniProt Protein Details:	Protein type:Membrane protein, integral; Membrane protein, multi-pass; Cell adhesion Cellular Component: Golgi apparatus; extracellular space; protein complex; cell surface; mitochondrion; intracellular membrane-bound organelle; endoplasmic reticulum; integral to membrane; caveola; lipid raft; membrane; apical part of cell; plasma membrane; intracellular; sarcolemma; external side of plasma membrane Molecular Function:low-density lipoprotein receptor activity; protein binding; transforming growth factor beta binding; low-density lipoprotein binding; high-density lipoprotein binding; lipid binding Biological Process: positive regulation of blood coagulation; positive regulation of interleukin-12 production; cGMP-mediated signaling; phagocytosis, recognition; pattern recognition receptor signaling pathway; negative regulation of transcription from RNA polymerase II promoter; signal transduction; fatty acid metabolic process; negative regulation of transcription factor import into nucleus; low density lipoprotein mediated signaling; sequestering of lipid; cell surface receptor linked signal transduction; positive regulation of MAPKKK cascade; transport; positive regulation of cell-matrix adhesion; regulation of transcription from RNA polymerase II promoter in response to oxidative stress; cell adhesion; long-chain fatty acid transport; receptor-mediated endocytosis; cholesterol transport; positive regulation of I-kappaB kinase/NF-kappaB cascade; negative regulation of systemic arterial blood pressure; positive regulation of interleukin-6 production; long-chain fatty acid metabolic process; positive regulation of tumor necrosis factor production; negative regulation of angiogenesis; positive regulation of peptidyl-tyrosine phosphorylation; defense response to Gram-positive bacterium; triacylglycerol metabolic process; response to mechanical stimulus; lipoprotein transport; fatty acid oxidation; positive regulation of phagocytosis, engulfment; plasma membrane long-chain fatty acid transport; nitric oxide mediated signal transduction; apoptotic cell clearance; fatty acid transport; positive regulation of cytokine secretion
UniProt Code:	Q08857
NCBI GenInfo Identifier:	729081
NCBI Gene ID:	12491
NCBI Accession:	Q08857.2
UniProt Related Accession:	Q08857
Molecular Weight:	52,698 Da
NCBI Full Name:	Platelet glycoprotein 4
NCBI Synonym Full Names:	CD36 antigen
NCBI Official Symbol:	Cd36
NCBI Official Synonym Symbols:	FAT; GPIV; Scarb3
NCBI Protein Information:	platelet glycoprotein 4; PAS-4; GPIIIB; PAS IV; glycoprotein IIIb; fatty acid translocase; platelet glycoprotein IV
UniProt Protein Name:	Platelet glycoprotein 4
UniProt Synonym Protein Names:	Glycoprotein IIIb; GPIIIB; PAS IV; PAS-4; Platelet glycoprotein IV; GPIV; CD_antigen: CD36
Protein Family:	Platelet glycoprotein
UniProt Gene Name:	Cd36
UniProt Entry Name:	CD36_MOUSE

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

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.