Human Amyloid Beta 40 / AB 1-40 ELISA Kit
Human Amyloid Beta 40 / AB 1-40 ELISA Kit - Information
The ELISA Genie Amyloid Beta 40 / AB 1-40 ELISA Kit can assay for Amyloid Beta 40 / AB 1-40 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How our Amyloid Beta 40 / AB 1-40 ELISA Kits Work?
The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.
At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Amyloid Beta 40 / AB 1-40 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.
Human Amyloid Beta 40 / AB 1-40 ELISA Kit - Data
Functions as a cell surface receptor and performs physiological functions on the surface of neurons relevant to neurite growth, neuronal adhesion and axonogenesis. Involved in cell mobility and transcription regulation through protein-protein interactions. Can promote transcription activation through binding to APBB1-KAT5 and inhibits Notch signaling through interaction with Numb. Couples to apoptosis-inducing pathways such as those mediated by G(O) and JIP. Inhibits G(o) alpha ATPase activity. Acts as a kinesin I membrane receptor, mediating the axonal transport of beta-secretase and presenilin 1. Involved in copper homeostasis/oxidative stress through copper ion reduction. In vitro, copper-metallated APP induces neuronal death directly or is potentiated through Cu2+-mediated low-density lipoprotein oxidation. Can regulate neurite outgrowth through binding to components of the extracellular matrix such as UFH and collagen I and IV. The splice isoforms that contain the BPTI domain possess protease inhibitor activity. Induces a AGER-dependent pathway that involves activation of p38 MAPK, resulting in internalization of amyloid-beta peptide and leading to mitochondrial dysfunction in cultured cortical neurons. Provides Cu2+ ions for GPC1 which are required for release of nitric oxide (NO) and subsequent degradation of the heparan sulfate chains on GPC1.; Beta-amyloid peptides are lipophilic metal chelators with metal-reducing activity. Bind transient metals such as copper, zinc and iron. In vitro, can reduce Cu2+ and Fe3+ to Cu+ and Fe2+, respectively. Beta-amyloid 42 is a more effective reductant than beta-amyloid 40. Beta-amyloid peptides bind to lipoproteins and apolipoproteins E and J in the CSF and to HDL particles in plasma, inhibiting metal-catalyzed oxidation of lipoproteins. Beta-APP42 may activate mononuclear phagocytes in the brain and elicit inflammatory responses. Promotes both tau aggregation and TPK II-mediated phosphorylation. Interaction with overexpressed HADH2 leads to oxidative stress and neurotoxicity. Also binds GPC1 in lipid rafts.; Appicans elicit adhesion of neural cells to the extracellular matrix and may regulate neurite outgrowth in the brain.; The gamma-CTF peptides as well as the caspase-cleaved peptides, including C31, are potent enhancers of neuronal apoptosis.; N-APP binds TNFRSF21 triggering caspase activation and degeneration of both neuronal cell bodies (via caspase-3) and axons (via caspase-6).
|Post-Translational Modification|| |
Proteolytically processed under normal cellular conditions. Cleavage either by alpha-secretase, beta-secretase or theta-secretase leads to generation and extracellular release of soluble APP peptides, S-APP-alpha and S-APP-beta, and the retention of corresponding membrane-anchored C-terminal fragments, C80, C83 and C99. Subsequent processing of C80 and C83 by gamma-secretase yields P3 peptides. This is the major secretory pathway and is non-amyloidogenic. Alternatively, presenilin/nicastrin-mediated gamma-secretase processing of C99 releases the amyloid beta proteins, amyloid-beta 40 (Abeta40) and amyloid-beta 42 (Abeta42), major components of amyloid plaques, and the cytotoxic C-terminal fragments, gamma-CTF(50), gamma-CTF(57) and gamma-CTF(59). Many other minor beta-amyloid peptides, beta-amyloid 1-X peptides, are found in cerebral spinal fluid (CSF) including the beta-amyloid X-15 peptides, produced from the cleavage by alpha-secretase and all terminating at Gln-686. Proteolytically cleaved by caspases during neuronal apoptosis. Cleavage at Asp-739 by either caspase-6, -8 or -9 results in the production of the neurotoxic C31 peptide and the increased production of beta-amyloid peptides. N- and O-glycosylated. O-glycosylation on Ser and Thr residues with core 1 or possibly core 8 glycans. Partial tyrosine glycosylation (Tyr-681) is found on some minor, short beta-amyloid peptides (beta-amyloid 1-15, 1-16, 1-17, 1-18, 1-19 and 1-20) but not found on beta-amyloid 38, beta-amyloid 40 nor on beta-amyloid 42. Modification on a tyrosine is unusual and is more prevelant in AD patients. Glycans had Neu5AcHex(Neu5Ac)HexNAc-O-Tyr, Neu5AcNeu5AcHex(Neu5Ac)HexNAc-O-Tyr and O-AcNeu5AcNeu5AcHex(Neu5Ac)HexNAc-O-Tyr structures, where O-Ac is O-acetylation of Neu5Ac. Neu5AcNeu5Ac is most likely Neu5Ac 2,8Neu5Ac linked. O-glycosylations in the vicinity of the cleavage sites may influence the proteolytic processing. Appicans are L-APP isoforms with O-linked chondroitin sulfate. Phosphorylation in the C-terminal on tyrosine, threonine and serine residues is neuron-specific. Phosphorylation can affect APP processing, neuronal differentiation and interaction with other proteins. Phosphorylated on Thr-743 in neuronal cells by Cdc5 kinase and Mapk10, in dividing cells by Cdc2 kinase in a cell-cycle dependent manner with maximal levels at the G2/M phase and, in vitro, by GSK-3-beta. The Thr-743 phosphorylated form causes a conformational change which reduces binding of Fe65 family members. Phosphorylation on Tyr-757 is required for SHC binding. Phosphorylated in the extracellular domain by casein kinases on both soluble and membrane-bound APP. This phosphorylation is inhibited by UFH. Extracellular binding and reduction of copper, results in a corresponding oxidation of Cys-144 and Cys-158, and the formation of a disulfide bond. In vitro, the APP-Cu+ complex in the presence of hydrogen peroxide results in an increased production of beta-amyloid-containing peptides.
A beta 40(Amyloid Beta 40)/Abeta 40/Beta-amyloid protein 40/AB(1-40)
|Detection method|| |
Sandwich ELISA Double Antibody
This immunoassay kit allows for the in vitro quantitative determination of AB40 concentrations in serum plasma and other biological fluids.
4'C for 6 months
Matrices listed below were spiked with certain level of AB40 and the recovery rates were calculated by comparing the measured value to the expected amount of Ab40 in samples.
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of AB40 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
For Research Use Only
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Human Amyloid Beta 40 / AB 1-40 ELISA Kit Protocol
The below protocol is a sample protocol for Human Amyloid Beta 40 / AB 1-40 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human Amyloid Beta 40 / AB 1-40 present in their sample.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 Â°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
|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 Â°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 37Â°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 37Â°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 Âµl of TMB substrate into each well, cover the plate and incubate at 37Â°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 Âµl 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.|
Human Amyloid Beta 40 / AB 1-40 ELISA Kit components
|ELISA Microplate(Dismountable)||8×12 strips||4°C for 6 months|
|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)|
Other materials and equipment required:The ELISA Genie Human Amyloid Beta 40 / AB 1-40 ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
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.
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.
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.
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.
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.
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.
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.
Human Amyloid Beta 40 / AB 1-40 ELISA Kit Protein Information
|UniProt Protein Function:||APP: a cell surface receptor that influences neurite growth, neuronal adhesion and axonogenesis. Cleaved by secretases to form a number of peptides, some of which bind to the acetyltransferase complex Fe65/TIP60 to promote transcriptional activation. The Abeta peptide is released from the cell, its extracellular deposition and accumulation form the main components of amyloid plaques in Alzheimer's disease. Mutations in this gene have been implicated in autosomal dominant Alzheimer disease and cerebroarterial amyloidosis. Can promote transcription activation through binding to Fe65-Tip60 and inhibits Notch signaling through interaction with Numb. Couples to apoptosis-inducing pathways such as those mediated by G(O) and JIP. Inhibits G(O) alpha ATPase activity. Acts as a kinesin I membrane receptor, mediating the axonal transport of beta-secretase and presenilin 1. Involved in copper homeostasis/oxidative stress through copper ion reduction. In vitro, copper-metallated APP induces neuronal death directly or is potentiated through Cu(2+)-mediated low-density lipoprotein oxidation. Can regulate neurite outgrowth through binding to components of the extracellular matrix such as heparin and collagen I and IV. Induces a RAGE-dependent pathway that activates p38 MAPK, resulting in internalization of amyloid-beta peptide and leading to mitochondrial dysfunction in cultured cortical neurons. Provides Cu(2+) ions for GPC1 which are required for release of nitric oxide (NO) and subsequent degradation of the heparan sulfate chains on GPC1. Binds, via its C-terminus, to the PID domain of several cytoplasmic proteins, including APBB family members, the APBA family, JIP1, SHC1 and, NUMB and DAB1. Binding to DAB1 inhibits its serine phosphorylation. Associates with microtubules in the presence of ATP and in a kinesin-dependent manner. Amyloid beta-42 binds nAChRA7 in hippocampal neurons. Beta-amyloid associates with HADH2. Soluble APP binds, via its N-terminal head, to FBLN1. Expressed in all fetal tissues examined with highest levels in brain, kidney, heart and spleen. Weak expression in liver. In adult brain, highest expression found in the frontal lobe of the cortex and in the anterior perisylvian cortex- opercular gyri. Moderate expression in the cerebellar cortex, the posterior perisylvian cortex-opercular gyri and the temporal associated cortex. Weak expression found in the striate, extra- striate and motor cortices. Expressed in cerebrospinal fluid, and plasma. 10 isoforms of the human protein are produced by alternative splicing. Isoform APP695 is the predominant form in neuronal tissue, isoform APP751 and isoform APP770 are widely expressed in non- neuronal cells. Isoform APP751 is the most abundant form in T-lymphocytes. Appican is expressed in astrocytes. The splice isoforms that contain the BPTI domain possess protease inhibitor activity. Belongs to the APP family.|
|UniProt Protein Details:|
Protein type:Receptor, misc.; Membrane protein, integral; Cell surface; Transcription factor; Apoptosis
Chromosomal Location of Human Ortholog: 21q21.3
Cellular Component: Golgi apparatus; extracellular space; cell surface; integral to plasma membrane; integral to membrane; coated pit; intercellular junction; cytosol; ER to Golgi transport vesicle; lipid raft; ciliary rootlet; perinuclear region of cytoplasm; nuclear envelope lumen; cytoplasm; synapse; dendritic shaft; neuromuscular junction; endosome; receptor complex; intracellular membrane-bound organelle; extracellular region; dendritic spine; axon; apical part of cell; plasma membrane; spindle midzone
Molecular Function:serine-type endopeptidase inhibitor activity; heparin binding; identical protein binding; protein binding; protease activator activity; enzyme binding; DNA binding; transition metal ion binding; PTB domain binding; acetylcholine receptor binding; receptor binding
Biological Process: extracellular matrix organization and biogenesis; adult locomotory behavior; mRNA polyadenylation; locomotory behavior; positive regulation of mitotic cell cycle; protein amino acid phosphorylation; regulation of translation; platelet degranulation; synaptic growth at neuromuscular junction; forebrain development; dendrite development; visual learning; collateral sprouting in the absence of injury; neuromuscular process controlling balance; cell adhesion; neurite development; cholesterol metabolic process; platelet activation; Notch signaling pathway; cellular copper ion homeostasis; regulation of epidermal growth factor receptor activity; axon cargo transport; mating behavior; regulation of multicellular organism growth; endocytosis; axon midline choice point recognition; smooth endoplasmic reticulum calcium ion homeostasis; negative regulation of neuron differentiation; neuron apoptosis; axonogenesis; suckling behavior; ionotropic glutamate receptor signaling pathway; regulation of synapse structure and activity; regulation of protein binding; innate immune response; positive regulation of transcription from RNA polymerase II promoter; response to oxidative stress; blood coagulation; neuron remodeling
Disease: Alzheimer Disease
|NCBI Summary:||This gene encodes a cell surface receptor and transmembrane precursor protein that is cleaved by secretases to form a number of peptides. Some of these peptides are secreted and can bind to the acetyltransferase complex APBB1/TIP60 to promote transcriptional activation, while others form the protein basis of the amyloid plaques found in the brains of patients with Alzheimer disease. In addition, two of the peptides are antimicrobial peptides, having been shown to have bacteriocidal and antifungal activities. Mutations in this gene have been implicated in autosomal dominant Alzheimer disease and cerebroarterial amyloidosis (cerebral amyloid angiopathy). Multiple transcript variants encoding several different isoforms have been found for this gene. [provided by RefSeq, Aug 2014]|
|NCBI GenInfo Identifier:||112927|
|NCBI Gene ID:||351|
|UniProt Secondary Accession:||P05067,P09000, P78438, Q13764, Q13778, Q13793, Q16011 B2R5V1, B4DII8, D3DSD1, D3DSD2, D3DSD3,|
|UniProt Related Accession:||P05067|
|NCBI Full Name:||Amyloid beta A4 protein|
|NCBI Synonym Full Names:||amyloid beta (A4) precursor protein|
|NCBI Official Symbol:||APP|
|NCBI Official Synonym Symbols:||AAA; AD1; PN2; ABPP; APPI; CVAP; ABETA; PN-II; CTFgamma|
|NCBI Protein Information:||amyloid beta A4 protein; preA4; protease nexin-II; peptidase nexin-II; beta-amyloid peptide; beta-amyloid peptide(1-40); beta-amyloid peptide(1-42); alzheimer disease amyloid protein; cerebral vascular amyloid peptide|
|UniProt Protein Name:||Amyloid beta A4 protein|
|UniProt Synonym Protein Names:||ABPP; APPI; APP; Alzheimer disease amyloid protein; Cerebral vascular amyloid peptide; CVAP; PreA4; Protease nexin-II; PN-II|
|Protein Family:||Amyloid beta A4 protein|
|UniProt Gene Name:||APP|
|UniProt Entry Name:||A4_HUMAN|