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Human Integrin Associated Protein/IAP ELISA Kit

SKU:
HUFI02567
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q08722
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich
Synonyms:
IAP, Protein MER6, Antigenic surface determinant protein OA3, Protein name:Leukocyte surface antigen CD47, antigen identified by monoclonal 1D8, CD47 antigen, Rh-related antigen, integrin-associated signal transducer, CD47 glycoprotein, CD47 molecule
Reactivity:
Human
€599
Frequently bought together:

Description

system_update_altDatasheetsystem_update_altMSDS
Product Name:Human Integrin Associated Protein / IAP ELISA Kit
Product Code:HUFI02567
Size:96 Assays
Alias:IAP, Protein MER6, Antigenic surface determinant protein OA3, Protein name:Leukocyte surface antigen CD47, antigen identified by monoclonal 1D8, CD47 antigen, Rh-related antigen, integrin-associated signal transducer, CD47 glycoprotein, CD47 molecule, integrin associated protein, Integrin-associated protein, leukocyte surface antigen CD47, MER6
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human IAP concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human IAP and the recovery rates were calculated by comparing the measured value to the expected amount of Human IAP in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)85-10191
EDTA plasma(n=5)85-10495
UFH plasma(n=5)85-10594
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human IAP and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)87-96%85-96%86-102%
EDTA plasma(n=5)88-96%86-100%85-96%
UFH plasma(n=5)80-97%81-100%80-93%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

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
UniprotQ08722
UniProt Protein Function:CD47: Has a role in both cell adhesion by acting as an adhesion receptor for THBS1 on platelets, and in the modulation of integrins. Plays an important role in memory formation and synaptic plasticity in the hippocampus. Receptor for SIRPA, binding to which prevents maturation of immature dendritic cells and inhibits cytokine production by mature dendritic cells. Interaction with SIRPG mediates cell-cell adhesion, enhances superantigen-dependent T-cell-mediated proliferation and costimulates T-cell activation. May play a role in membrane transport and/or integrin dependent signal transduction. May prevent premature elimination of red blood cells. May be involved in membrane permeability changes induced following virus infection. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:Membrane protein, integral; Membrane protein, multi-pass; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 3q13.1-q13.2

Cellular Component: integral to plasma membrane; plasma membrane

Molecular Function:protein binding

Biological Process: integrin-mediated signaling pathway; extracellular matrix organization and biogenesis; response to bacterium; positive regulation of phagocytosis; positive regulation of cell proliferation; positive regulation of cell-cell adhesion; positive regulation of T cell activation; opsonization; blood coagulation; cell adhesion; leukocyte migration; positive regulation of inflammatory response

NCBI Summary:This gene encodes a membrane protein, which is involved in the increase in intracellular calcium concentration that occurs upon cell adhesion to extracellular matrix. The encoded protein is also a receptor for the C-terminal cell binding domain of thrombospondin, and it may play a role in membrane transport and signal transduction. This gene has broad tissue distribution, and is reduced in expression on Rh erythrocytes. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Jul 2010]
UniProt Code:Q08722
NCBI GenInfo Identifier:1171879
NCBI Gene ID:961
NCBI Accession:Q08722.1
UniProt Related Accession:Q08722
Molecular Weight:
NCBI Full Name:Leukocyte surface antigen CD47
NCBI Synonym Full Names:CD47 molecule
NCBI Official Symbol:CD47  
NCBI Official Synonym Symbols:IAP; OA3; MER6  
NCBI Protein Information:leukocyte surface antigen CD47
UniProt Protein Name:Leukocyte surface antigen CD47
UniProt Synonym Protein Names:Antigenic surface determinant protein OA3; Integrin-associated protein; IAP; Protein MER6; CD_antigen: CD47
Protein Family:IgE-binding protein
UniProt Gene Name:CD47  
UniProt Entry Name:CD47_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.

StepProtocol
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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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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