Rat Platelet glycoprotein 4 (Cd36) ELISA Kit

Product Name:	Rat Platelet glycoprotein 4 (Cd36) ELISA Kit
SKU:	RTEB0397
Size:	96T
Target:	Rat Platelet glycoprotein 4 (Cd36)
Synonyms:	Adipocyte membrane protein, Fatty acid translocase, Fatty acid transport protein, Glycoprotein IIIb, PAS IV, PAS-4, Platelet glycoprotein IV, GPIIIB, GPIV, CD36, Fat
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.312-20ng/mL
Sensitivity:	0.158ng/mL

Intra CV:	Provided with the Kit
Inter CV:	Provided with the Kit
Linearity:	Provided with the Kit
Recovery:	Provided with the Kit
Function:	Multifunctional glycoprotein that acts as receptor for a broad range of ligands. Ligands can be of proteinaceous nature like thrombospondin, fibronectin, collagen or amyloid-beta as well as of lipidic nature such as oxidized low-density lipoprotein (oxLDL), anionic phospholipids, long-chain fatty acids and bacterial diacylated lipopeptides. They are generally multivalent and can therefore engage multiple receptors simultaneously, the resulting formation of CD36 clusters initiates signal transduction and internalization of receptor-ligand complexes. The dependency on coreceptor signaling is strongly ligand specific. Cellular responses to these ligands are involved in angiogenesis, inflammatory response, fatty acid metabolism, taste and dietary fat processing in the intestine (By similarity) (PubMed:8320718). Binds long-chain fatty acids and facilitates their transport into cells, thus participating in muscle lipid utilization, adipose energy storage, and gut fat absorption (By similarity) (PubMed:8320718). In the small intestine, plays a role in proximal absorption of dietary fatty acid and cholesterol for optimal chylomicron formation, possibly through the activation of MAPK1/3 (ERK1/2) signaling pathway (By similarity) (PubMed:16276419, PubMed:21610069). Involved in oral fat perception and preferences (By similarity) (PubMed:16276419). Detection into the tongue of long-chain fatty acids leads to a rapid and sustained rise in flux and protein content of pancreatobiliary secretions (By similarity) (PubMed:16276419). In taste receptor cells, mediates the induction of an increase in intracellulare calcium levels by long-chain fatty acids, leading to the activation of the gustatory neurons in the nucleus of the solitary tract (By similarity). Important factor in both ventromedial hypothalamus neuronal sensing of long-chain fatty acid and the regulation of energy and glucose homeostasis (By similarity) (PubMed:23557700). Receptor for thombospondins, THBS1 and THBS2, mediating their antiangiogenic effects (By similarity). As a coreceptor for TLR4:TLR6 heterodimer, promotes inflammation in monocytes/macrophages. Upon ligand binding, such as oxLDL or amyloid-beta 42, interacts with the heterodimer TLR4:TLR6, the complex is internalized and triggers inflammatory response, leading to NF-kappa-B-dependent production of CXCL1, CXCL2 and CCL9 cytokines, via MYD88 signaling pathway, and CCL5 cytokine, via TICAM1 signaling pathway, as well as IL1B secretion, through the priming and activation of the NLRP3 inflammasome. Selective and nonredundant sensor of microbial diacylated lipopeptide that signal via TLR2:TLR6 heterodimer, this cluster triggers signaling from the cell surface, leading to the NF-kappa-B-dependent production of TNF, via MYD88 signaling pathway and subsequently is targeted to the Golgi in a lipid-raft dependent pathway.

Uniprot:	Q07969
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Platelet glycoprotein 4
Sub Unit:	Interacts with THBS1 and THBS2; the interactions mediate the THBS antiangiogenic activity. Upon interaction with a ligand, such as oxidized low-density lipoprotein (oxLDL) or amyloid-beta 42, rapidly forms a complex with TLR4 and TLR6; the complex is internalized and triggers an inflammatory signal. Through its C-terminus, interacts with PTK2, PXN and LYN, but not with SRC. LYN kinase activity is required for facilitating TLR4:TLR6 heterodimerization and signal initiation. Upon interaction with ligands such as diacylated lipopeptides, interacts with the TLR2:TLR6 heterodimer (By similarity). Interacts with CD9, CD81, FCER1G, ITGB2 and/or ITGB2; forming a membrane heteromeric complex required for the internalization of CD36 and its ligands.
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: Binds to collagen, thrombospondin, anionic phospholipids and oxidized low-density lipoprotein (oxLDL). 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 (By similarity). Receptor for thombospondins, THBS1 AND THBS2, mediating their antiangiogenic effects (By similarity). As a coreceptor for TLR4-TLR6, promotes inflammation in monocytes/macrophages. Upon ligand binding, such as oxLDL or amyloid-beta 42 binding, rapidly induces the formation of a heterodimer of TLR4 and TLR6, which is internalized and triggers inflammatory signals, leading to the NF-kappa-B-dependent production of CXCL1, CXCL2 and CCL9 cytokines, via MYD88 signaling pathway, and CCL5 cytokine, via TICAM1 signaling pathway, as well as IL1B secretion
UniProt Protein Details:	Protein type:Cell adhesion; Membrane protein, integral; Membrane protein, multi-pass Chromosomal Location of Human Ortholog: 4q11 Cellular Component: apical part of cell; apical plasma membrane; brush border membrane; caveola; cell surface; endoplasmic reticulum; external side of plasma membrane; extracellular space; Golgi apparatus; integral component of membrane; intracellular; intracellular membrane-bound organelle; membrane; membrane raft; mitochondrion; plasma membrane; protein complex; receptor complex; sarcolemma Molecular Function:high-density lipoprotein particle binding; lipid binding; low-density lipoprotein particle binding; low-density lipoprotein receptor activity; protein binding; transforming growth factor beta binding Biological Process: apoptotic cell clearance; cell surface receptor signaling pathway; cellular response to insulin stimulus; cGMP-mediated signaling; cholesterol transport; defense response to Gram-positive bacterium; eating behavior; elevation of cytosolic calcium ion concentration; fatty acid metabolic process; fatty acid oxidation; fatty acid transport; glucose homeostasis; gut development; immune response; interleukin-1 beta secretion; intestinal absorption; intestinal cholesterol absorption; lipid storage; lipoprotein transport; long-chain fatty acid metabolic process; long-chain fatty acid transport; low density lipoprotein mediated signaling; maternal placenta development; negative regulation of angiogenesis; negative regulation of systemic arterial blood pressure; negative regulation of transcription factor import into nucleus; negative regulation of transcription from RNA polymerase II promoter; nitric oxide mediated signal transduction; phagocytosis, recognition; positive regulation of blood coagulation; positive regulation of cell-matrix adhesion; positive regulation of cytokine secretion; positive regulation of I-kappaB kinase/NF-kappaB signaling; positive regulation of interleukin-12 production; positive regulation of interleukin-6 production; positive regulation of MAPK cascade; positive regulation of NF-kappaB transcription factor activity; positive regulation of nitric oxide biosynthetic process; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of phagocytosis; positive regulation of phagocytosis, engulfment; positive regulation of tumor necrosis factor production; receptor internalization; regulation of protein heterodimerization activity; regulation of toll-like receptor signaling pathway; response to activity; response to drug; response to estradiol; response to lipid; response to mechanical stimulus; response to nutrient; sensory perception of taste; triacylglycerol metabolic process; triglyceride transport
NCBI Summary:	fatty acid translocase; involved in long-chain fatty acid (LCFA) transport; important in fatty acid metabolism and insulin function [RGD, Feb 2006]
UniProt Code:	Q07969
NCBI GenInfo Identifier:	146345388
NCBI Gene ID:	29184
NCBI Accession:	Q07969.3
UniProt Secondary Accession:	Q07969,Q925W0,
UniProt Related Accession:	Q07969
Molecular Weight:	52,731 Da
NCBI Full Name:	Platelet glycoprotein 4
NCBI Synonym Full Names:	CD36 molecule
NCBI Official Symbol:	Cd36
NCBI Official Synonym Symbols:	Fat; RGD1562323
NCBI Protein Information:	platelet glycoprotein 4
UniProt Protein Name:	Platelet glycoprotein 4
UniProt Synonym Protein Names:	Adipocyte membrane protein; Fatty acid translocase; Fatty acid transport protein; Glycoprotein IIIb; GPIIIB; PAS IV; PAS-4; Platelet glycoprotein IV; GPIV; CD_antigen: CD36
Protein Family:	Glycoprotein
UniProt Gene Name:	Cd36

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.

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Rat Platelet glycoprotein 4 (Cd36) ELISA Kit (RTEB0397)

Description