Human Platelet glycoprotein 4 (CD36) ELISA Kit

Product Name:	Human Platelet glycoprotein 4 (CD36) ELISA Kit
SKU:	HUEB0795
Size:	96T
Target:	Human Platelet glycoprotein 4 (CD36)
Synonyms:	Fatty acid translocase, Glycoprotein IIIb, Leukocyte differentiation antigen CD36, PAS IV, PAS-4, Platelet collagen receptor, Platelet glycoprotein IV, Thrombospondin receptor, FAT, GPIIIB, GPIV, CD36, GP3B, GP4
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.078ng/mL

Intra CV:

3.2%

Inter CV:

7.1%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	84-93%	96-106%	100-108%	108-117%
EDTA Plasma(N=5)	85-95%	80-93%	90-100%	95-105%
Heparin Plasma(N=5)	104-116%	81-93%	102-111%	93-103%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	101	95-107
Plasma	103	97-109

Function:

(Microbial infection) Directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes and the internalization of particles independently of TLR signaling.

Uniprot:	P16671
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Platelet glycoprotein 4
Sub Unit:	(Microbial infection) Binds to Plasmodium falciparum EMP1.
Research Area:	Immunology
Subcellular Location:	Cell membrane Multi-pass membrane protein Membrane raft Golgi apparatus Apical cell membrane Upon ligand-binding, internalized through dynamin-dependent endocytosis.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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, multi-pass; Membrane protein, integral; Cell adhesion Chromosomal Location of Human Ortholog: 7q11.2 Cellular Component: Golgi apparatus; extracellular space; platelet alpha granule membrane; cell surface; membrane; integral to plasma membrane; plasma membrane; phagocytic vesicle; lipid raft; external side of plasma membrane Molecular Function:low-density lipoprotein receptor activity; transforming growth factor beta binding; low-density lipoprotein binding; lipid binding; high-density lipoprotein binding Biological Process: positive regulation of blood coagulation; cGMP-mediated signaling; positive regulation of interleukin-12 production; phagocytosis, recognition; negative regulation of transcription from RNA polymerase II promoter; cellular lipid metabolic process; negative regulation of transcription factor import into nucleus; low density lipoprotein mediated signaling; sequestering of lipid; antigen processing and presentation of peptide antigen via MHC class I; cell surface receptor linked signal transduction; platelet degranulation; positive regulation of MAPKKK cascade; antigen processing and presentation of exogenous peptide antigen via MHC class I; positive regulation of cell-matrix adhesion; cell adhesion; toll-like receptor 4 signaling pathway; receptor-mediated endocytosis; platelet activation; positive regulation of I-kappaB kinase/NF-kappaB cascade; cholesterol transport; positive regulation of interleukin-6 production; antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent; positive regulation of tumor necrosis factor production; toll-like receptor 2 signaling pathway; MyD88-dependent toll-like receptor signaling pathway; defense response to Gram-positive bacterium; positive regulation of peptidyl-tyrosine phosphorylation; lipoprotein transport; toll-like receptor signaling pathway; innate immune response; lipid metabolic process; blood coagulation; positive regulation of phagocytosis, engulfment; plasma membrane long-chain fatty acid transport; nitric oxide mediated signal transduction; apoptotic cell clearance Disease: Platelet Glycoprotein Iv Deficiency; Coronary Heart Disease, Susceptibility To, 7; Malaria, Susceptibility To
NCBI Summary:	The protein encoded by this gene is the fourth major glycoprotein of the platelet surface and serves as a receptor for thrombospondin in platelets and various cell lines. Since thrombospondins are widely distributed proteins involved in a variety of adhesive processes, this protein may have important functions as a cell adhesion molecule. It binds to collagen, thrombospondin, anionic phospholipids and oxidized LDL. It directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes and it binds long chain fatty acids and may function in the transport and/or as a regulator of fatty acid transport. Mutations in this gene cause platelet glycoprotein deficiency. Multiple alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Feb 2014]
UniProt Code:	P16671
NCBI GenInfo Identifier:	115982
NCBI Gene ID:	948
NCBI Accession:	P16671.2
UniProt Secondary Accession:	P16671,Q13966, Q16093, Q8TCV7, Q9BPZ8, Q9BQC2, Q9BZM8 Q9BZN3, D9IX66, D9IX67, D9IX68, D9IX69,
UniProt Related Accession:	P16671
Molecular Weight:	472
NCBI Full Name:	Platelet glycoprotein 4
NCBI Synonym Full Names:	CD36 molecule (thrombospondin receptor)
NCBI Official Symbol:	CD36
NCBI Official Synonym Symbols:	FAT; GP4; GP3B; GPIV; CHDS7; PASIV; SCARB3; BDPLT10
NCBI Protein Information:	platelet glycoprotein 4; GPIIIB; PAS IV; PAS-4 protein; glycoprotein IIIb; cluster determinant 36; fatty acid translocase; platelet glycoprotein IV; scavenger receptor class B, member 3; leukocyte differentiation antigen CD36; CD36 antigen (collagen type
UniProt Protein Name:	Platelet glycoprotein 4
UniProt Synonym Protein Names:	Fatty acid translocase; FAT; Glycoprotein IIIb; GPIIIB; Leukocyte differentiation antigen CD36; PAS IV; PAS-4; Platelet collagen receptor; Platelet glycoprotein IV; GPIV; Thrombospondin receptor; CD_antigen: CD36
Protein Family:	Fat body protein
UniProt Gene Name:	CD36
UniProt Entry Name:	CD36_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 GP4 / CD36 ELISA Kit	Anti-CD36 Antibody (CAB1470)
Human GP4/CD36 (Platelet Membrane Glycoprotein IV) CLIA Kit (HUES00613)	Anti-CD36 Antibody (CAB14714)
Human GP4/CD36 (Platelet Membrane Glycoprotein IV) ELISA Kit (HUES02146)	Anti-CD36 Antibody (CAB17339)
	Anti-CD36 Antibody (CAB17340)
	Anti-CD36 Antibody (CAB19016)