Product Name:	Human CD51 / Integrin alpha V ELISA Kit
Product Code:	HUFI00596
Size:	96 Assays
Alias:	ITGAV, Integrin alpha-V, CD51, MSK8, Vitronectin Receptor alpha, VNRA, antigen identified by monoclonal L230, CD51 antigen, integrin, alpha V, vitronectin receptor, alpha polypeptide, antigen CD51, Vitronectin receptor subunit alpha
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human ITGAV 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 Human ITGAV and the recovery rates were calculated by comparing the measured value to the expected amount of Human ITGAV in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-103	94
EDTA plasma(n=5)	87-104	99
UFH plasma(n=5)	88-101	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ITGAV 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)	85-102%	85-105%	90-97%
EDTA plasma(n=5)	85-93%	82-98%	87-99%
UFH plasma(n=5)	81-99%	80-96%	85-93%

CV(%):

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

P06756

UniProt Protein Function:	ITGAV: The alpha-V integrins are receptors for vitronectin, cytotactin, fibronectin, fibrinogen, laminin, matrix metalloproteinase-2, osteopontin, osteomodulin, prothrombin, thrombospondin and vWF. They recognize the sequence R-G-D in a wide array of ligands. In case of HIV-1 infection, the interaction with extracellular viral Tat protein seems to enhance angiogenesis in Kaposi's sarcoma lesions. Belongs to the integrin alpha chain family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell adhesion; Membrane protein, integral; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 2q31-q32 Cellular Component: filopodium membrane; focal adhesion; cell surface; membrane; integral to plasma membrane; microvillus membrane; plasma membrane; phagocytic vesicle; integrin complex; external side of plasma membrane Molecular Function:voltage-gated calcium channel activity; viral receptor activity; opsonin binding; protein binding; insulin-like growth factor I binding; protein kinase C binding; protease binding; extracellular matrix binding; transforming growth factor beta binding; metal ion binding; fibronectin binding Biological Process: negative regulation of lipid transport; axon guidance; entry of virus into host cell; extracellular matrix organization and biogenesis; positive regulation of cell adhesion; cell-matrix adhesion; negative chemotaxis; entry of symbiont into host cell by promotion of host phagocytosis; regulation of phagocytosis; antigen processing and presentation of peptide antigen via MHC class I; positive regulation of cell proliferation; antigen processing and presentation of exogenous peptide antigen via MHC class I; angiogenesis; cell adhesion; cell growth; integrin-mediated signaling pathway; cell migration; negative regulation of lipoprotein metabolic process; antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent; cell-substrate adhesion; heterotypic cell-cell adhesion; negative regulation of low-density lipoprotein receptor biosynthetic process; positive regulation of osteoblast proliferation; blood coagulation; vascular endothelial growth factor receptor signaling pathway; leukocyte migration; apoptotic cell clearance; positive regulation of cell migration
NCBI Summary:	This gene encodes a protein that is a member of the integrin superfamily. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. This protein undergoes post-translational cleavage to yield disulfide-linked heavy and light chains that combine with multiple integrin beta chains to form different integrins. This protein has been shown to heterodimerize with beta 1, beta 3, beta 5, beta 6, and beta 8; the heterodimer of alpha v and beta 3 is the Vitronectin receptor. This protein interacts with several extracellular matrix proteins to mediate cell adhesion and may play a role in cell migration. It is proposed that this protein may regulate angiogenesis and cancer progression. Alternative splicing results in multiple transcript variants that encode different protein isoforms. Note that the integrin alpha 5 and integrin alpha V chains are produced by distinct genes. [provided by RefSeq, Jan 2015]
UniProt Code:	P06756
NCBI GenInfo Identifier:	143811408
NCBI Gene ID:	3685
NCBI Accession:	P06756.2
UniProt Secondary Accession:	P06756,Q53SK4, Q59EB7, Q6LD15, A0AV67, B0LPF4, B7Z883 B7ZLX0, D3DPG8, E7EWZ6,
UniProt Related Accession:	P06756
Molecular Weight:	1048
NCBI Full Name:	Integrin alpha-V
NCBI Synonym Full Names:	integrin, alpha V
NCBI Official Symbol:	ITGAV
NCBI Official Synonym Symbols:	CD51; MSK8; VNRA; VTNR
NCBI Protein Information:	integrin alpha-V; integrin alphaVbeta3; vitronectin receptor subunit alpha; antigen identified by monoclonal antibody L230; integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)
UniProt Protein Name:	Integrin alpha-V
UniProt Synonym Protein Names:	Vitronectin receptor subunit alpha; CD_antigen: CD51Cleaved into the following 2 chains:Integrin alpha-V heavy chain; Integrin alpha-V light chain
Protein Family:	Integrin
UniProt Gene Name:	ITGAV
UniProt Entry Name:	ITAV_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
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.

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Human CD51 / Integrin alpha V ELISA Kit

Description