Rat GP4/CD36 ELISA Kit (RTFI00009)

Product Name:	Rat GP4/CD36 ELISA Kit
Product Code:	RTFI00009
Size:	96 Assays
Target:	Rat GP4/CD36
Alias:	GP4, 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
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
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 Rat GP4/CD36 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat GP4/CD36 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-102	94
EDTA plasma(n=5)	93-104	99
UFH plasma(n=5)	86-101	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat 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)	86-100%	91-104%	89-104%
EDTA plasma(n=5)	86-96%	88-101%	86-96%
UFH plasma(n=5)	83-90%	81-100%	85-90%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

Q07969

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

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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.