Mouse Amyloid beta A4 protein (App) ELISA Kit

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SKU:
MOEB0675
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P12023
Range:
78-5000 pg/mL
ELISA Type:
Sandwich
Synonyms:
Amyloid-beta A4 protein, ABPP, APP
Reactivity:
Mouse
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Description

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Product Name:Mouse Amyloid beta A4 protein (App) ELISA Kit
Product Code:MOEB0675
Alias:Amyloid beta A4 protein, ABPP, APP, Alzheimer disease amyloid A4 protein homolog, Amyloidogenic glycoprotein, AG, App
Uniprot:P12023
Reactivity:Mouse
Range:78-5000 pg/mL
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
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:Cell surface; Membrane protein, integral; Apoptosis; Transcription factor; Receptor, misc.

Cellular Component: Golgi apparatus; extracellular space; cell surface; smooth endoplasmic reticulum; integral to membrane; terminal button; coated pit; intercellular junction; ER to Golgi transport vesicle; lipid raft; ciliary rootlet; membrane; perinuclear region of cytoplasm; nuclear envelope lumen; cytoplasm; cytoplasmic vesicle; intracellular; neuromuscular junction; receptor complex; endosome; neuron projection; intracellular membrane-bound organelle; cell; apical part of cell; axon; plasma membrane; spindle midzone

Molecular Function:serine-type endopeptidase inhibitor activity; heparin binding; identical protein binding; protease activator activity; protease inhibitor activity; metal ion binding; PTB domain binding; acetylcholine receptor binding; protein binding; enzyme binding; DNA binding; transition metal ion binding; receptor binding

Biological Process: extracellular matrix organization and biogenesis; apoptosis; adult locomotory behavior; negative regulation of peptidase activity; locomotory behavior; mRNA polyadenylation; positive regulation of mitotic cell cycle; protein amino acid phosphorylation; regulation of translation; synaptic growth at neuromuscular junction; forebrain development; dendrite development; visual learning; collateral sprouting in the absence of injury; neuromuscular process controlling balance; cell adhesion; protein homooligomerization; neurite development; cholesterol metabolic process; nervous system development; Notch signaling pathway; cellular copper ion homeostasis; regulation of epidermal growth factor receptor activity; mating behavior; axon cargo transport; regulation of multicellular organism growth; endocytosis; axon midline choice point recognition; smooth endoplasmic reticulum calcium ion homeostasis; negative regulation of neuron differentiation; neuron apoptosis; suckling behavior; axonogenesis; regulation of gene expression; ionotropic glutamate receptor signaling pathway; regulation of synapse structure and activity; regulation of protein binding; positive regulation of transcription from RNA polymerase II promoter; response to oxidative stress; neuron remodeling

UniProt Code:P12023
NCBI GenInfo Identifier:311893401
NCBI Gene ID:11820
NCBI Accession:NP_001185752.1
UniProt Secondary Accession:P12023,P97487, P97942, Q99K32,
UniProt Related Accession:P12023
Molecular Weight:Predicted Molecular Mass: 12.5kDaAccurate Molecular Mass: 12kDa as determined by SDS-PAGE reducing conditions.
NCBI Full Name:amyloid beta A4 protein isoform 1
NCBI Synonym Full Names:amyloid beta (A4) precursor protein
NCBI Official Symbol:App  
NCBI Official Synonym Symbols:Ag; Abpp; Adap; Cvap; Abeta; betaApp; E030013M08Rik  
NCBI Protein Information:amyloid beta A4 protein; appican; protease nexin II; amyloidogenic glycoprotein; alzheimer disease amyloid A4 protein homolog
UniProt Protein Name:Amyloid beta A4 protein
UniProt Synonym Protein Names:ABPP; APP; Alzheimer disease amyloid A4 protein homolog; Amyloidogenic glycoprotein; AGCleaved into the following 14 chains:N-APPSoluble APP-alpha; S-APP-alphaSoluble APP-beta; S-APP-betaC99; Alternative name(s):; APP-C99Beta-amyloid protein 42; Alternative name(s):; Beta-APP42Beta-amyloid protein 40; Alternative name(s):; Beta-APP40C83P3(42)P3(40)C80Gamma-secretase C-terminal fragment 59; Alternative name(s):; APP-C59; Amyloid intracellular domain 59; AID(59); Gamma-CTF(59)Gamma-secretase C-terminal fragment 57; Alternative name(s):; APP-C57; Amyloid intracellular domain 57; AID(57); Gamma-CTF(57)Gamma-secretase C-terminal fragment 50; Alternative name(s):; Amyloid intracellular domain 50; AID(50); Gamma-CTF(50)
Protein Family:Amyloid beta A4 protein
UniProt Gene Name:App  
UniProt Entry Name:A4_MOUSE
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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