Human APP / Amyloid Precursor Protein ELISA Kit PicoKine™

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SKU	EK0658
Size	96wells/kit, with removable strips.
Reactivity	Human
Sample Type	cell culture supernates, cell lysates, serum and plasma (heparin, EDTA).
Sample Volume	100ul per well
Sensitivity	<100pg/ml
Assay Range	312pg/ml-20,000pg/ml

• Overview
• Assay Details
• Target Info
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Overview

Product Name	Human APP / Amyloid Precursor Protein ELISA Kit PicoKine™
SKU/Catalog Number	EK0658
Storage & Handling	Store at 4°C for 6 months, at -20°C for 12 months. Avoid multiple freeze-thaw cycles(Shipped with wet ice.)
Size	96wells/kit, with removable strips.
Description	Sandwich High Sensitivity ELISA kit for Quantitative Detection of Human APP. 96wells/kit, with removable strips.
Cite This Product	Human APP / Amyloid Precursor Protein ELISA Kit PicoKine™ (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK0658)
Sample Type	cell culture supernates, cell lysates, serum and plasma (heparin, EDTA). Anticoagulant(s): heparin or EDTA *The recommended anticoagulants are proven to not block the antibody binding sites on the target antigen. Please do not collect blood sample with other anticoagulants that are not specified above or contact us to check for feasibility.
Sensitivity	<100pg/ml
Assay Range	312pg/ml-20,000pg/ml
Immunogen	Expression system for standard: NSO; Immunogen sequence: L18-L688
Reactivity	Human
Cross Reactivity	There is no detectable cross-reactivity with other relevant proteins.
Antibody Clonalities	Capture Antibody | Detection Antibody: monoclonal antibody from mouse|polyclonal antibody from goat

Assay Details

Kit Components

Catalog number	Description	Quantity
EK0658-CAP	96-well plate precoated with anti-Human APP antibody	1
EK0658-ST	lyophilized recombinant Human APP standard	20ng/tube
EK0658-DA	biotinylated anti-Human APP antibody	130ul
AR1103	Avidin-Biotin-Peroxidase Complex(ABC)	130ul
AR1106-1	sample diluent buffer	30ml
AR1106-2	antibody diluent buffer	12ml
AR1106-3	ABC diluent buffer	12ml
AR1104	TMB color developing agent	10ml
AR1105	TMB stop solution	10ml
PLA-SEA	Adhesive cover	4

*Why there is no wash buffer? Our Avidin-Biotin-Peroxidase Diluent contains the detergent (TWEEN) normally present in other companies' ELISA kits. This saves you the step of having to wash with the special wash buffer and achieve similar or better signal to noise ratio. The wash can use regular wash buffers (PBS, TBS etc.) commonly found in labs.

Materials Required But Not Provided

• Microplate reader in standard size.
• Automated plate washer.
• Adjustable pipettes and pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection.
• Clean tubes and Eppendorf tubes.
• Washing buffer (neutral PBS or TBS).
• Preparation of 0.01M TBS: Add 1.2g Tris, 8.5g NaCl; 450μl of purified acetic acid or 700μl of concentrated hydrochloric acid to 1000ml H2
• Preparation of 0.01 M PBS: Add 8.5g sodium chloride, 1.4g Na O and adjust pH to 7.2-7.

Typical Data Obtained from Human APP / Amyloid Precursor Protein ELISA Kit PicoKine™

(TMB reaction incubate at 37°C for 15-25min)

Concentration(pg/ml)	0	312	625	200000000	20000000	2000000	200000	20000
O.D.	0.130	0.181	0.221	0.323	0.506	0.788	1.248	1.854

Intra/Inter Assay Precision

	Intra-Assay Precision			Inter-Assay Precision
Sample	1	2	3	1	2	3
n	16	16	16	24	24	24
Mean(pg/ml)	775	2619	9426	755	2868	9446
Standard deviation	50.37	188.56	377.04	54.36	226.57	510.08
CV(%)	6.5%	7.2%	4%	7.2%	7.9%	5.4%

Reproducibility

Three samples with differing target protein concentrations were assayed using four different lots to measure the CV% lot to lot variance.

Reproducibility

To assay reproducibility, three samples with differing target protein concentrations were assayed using four different lots.

Lots	Lot1 (pg/ml)	Lot2 (pg/ml)	Lot3 (pg/ml)	Lot4 (pg/ml)	Mean (pg/ml)	Standard Deviation	CV (%)
Sample 1	775	742	738	700	738	26.58	3.6%
Sample 2	2619	3033	2635	3053	2835	208.19	7.3%
Sample 3	9426	9494	8945	9504	9342	231.3	2.4%

*number of samples for each test n=16.

*The typical data is obtained from Boster's internal QC result and for reference only. It may differ from the lab test results of the end users. It is more important that the user's lab test results reflect the same linearity demonstrated in the typical data than achieving exactly the same O.D. values.

Target Info

Protein Target Info (Source: Uniprot.org)

Uniprot Id	P05067
Gene Name	APP
Protein Name	Amyloid beta A4 protein
Tissue Specificity	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. 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. .
Alternative Names	Amyloid beta A4 protein;ABPP;APPI;APP;Alzheimer disease amyloid protein;Cerebral vascular amyloid peptide;CVAP;PreA4;Protease nexin-II;PN-II;N-APP;Soluble APP-alpha;S-APP-alpha;Soluble APP-beta;S-APP-beta;C99;Beta-amyloid protein 42;Beta-APP42;Beta-amyloid protein 40;Beta-APP40;C83;P3(42);P3(40);C80;Gamma-secretase C-terminal fragment 59;Amyloid intracellular domain 59;AICD-59;AID(59);Gamma-CTF(59);Gamma-secretase C-terminal fragment 57;Amyloid intracellular domain 57;AICD-57;AID(57);Gamma-CTF(57);Gamma-secretase C-terminal fragment 50;Amyloid intracellular domain 50;AICD-50;AID(50);Gamma-CTF(50);C31;APP;A4, AD1;
Subcellular Localization	Membrane; Single-pass type I membrane protein. Membrane, clathrin-coated pit. Cell surface protein that rapidly becomes internalized via clathrin-coated pits. During maturation, the immature APP (N-glycosylated in the endoplasmic reticulum) moves to the Golgi complex where complete maturation occurs (O-glycosylated and sulfated). After alpha-secretase cleavage, soluble APP is released into the extracellular space and the C-terminal is internalized to endosomes and lysosomes. Some APP accumulates in secretory transport vesicles leaving the late Golgi compartment and returns to the cell surface. Gamma-CTF(59) peptide is located to both the cytoplasm and nuclei of neurons. It can be translocated to the nucleus through association with APBB1 (Fe65). Beta-APP42 associates with FRPL1 at the cell surface and the complex is then rapidly internalized. APP sorts to the basolateral surface in epithelial cells. During neuronal differentiation, the Thr-743 phosphorylated form is located mainly in growth cones, moderately in neurites and sparingly in the cell body. Casein kinase phosphorylation can occur either at the cell surface or within a post-Golgi compartment. Associates with GPC1 in perinuclear compartments. Colocalizes with SORL1 in a vesicular pattern in cytoplasm and perinuclear regions.
Molecular Weight	86943 MW

*if product is indicated to react with multiple species, protein info is based on the human gene.

Ontology

Protein Function	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 (By similarity). 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. 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 Cu(2+) ions for GPC1 which are required for release of nitric oxide (NO) and subsequent degradation of the heparan sulfate chains on GPC1. .
Research Areas	Human *You can search these to find other products in these research areas.
Background	Amyloid precursor protein(APP) is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation, neural plasticity and iron export. APP is best known and most commonly studied as the precursor molecule whose proteolysis generates beta amyloid(Abeta), a 39- to 42-amino acid peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients. APP undergoes posttranslational proteolytic processing by alpha-, beta-, and gamma-secretases. Alpha-secretase generates soluble amyloid protein, while beta- and gamma-secretases generate APP components with amyloidogenic features. These 2 processing pathways are mutually exclusive.

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Customer Q&As

• Q: I found bubbles in the wells of my ELISA kit. Is this normal or will it have an impact on the performance of the kit?
  A: The bubbles in the wells is an uncommon issue, but it would not affect the results of your experiment.
  We suggest to wash the plate to remove the bubbles as suggested in the protocol below prior to the assay:

  1. Wash the plate 3 times with the 1X wash buffer.
  2. Discard the liquid in the wells into an appropriate waste receptacle. Then, invert the plate on the benchtop onto a paper towel and tap the plate to gently blot any remaining liquid. It is recommended that the wells are not allowed to completely dry at any time.
  3. Add 300 µl of the 1x wash buffer to each assay well. (For cleaner background incubate for 60 seconds between each wash).
  4. Repeat steps a-b 2 additional times.
• Q: There are precipitates in the sample diluent buffer. What is it?
  A: The precipitates are fatty substances contained in BSA. It would not affect the performance of the kit. If you want to confirm this, please contact Boster Support.
• Q: I left this product out in ambient temperatures for around 5 days, will the kit be stable?
  A: Our kits should still be stable after being in ambient temperatures for around 5 days. It would not affect the performance of the kit. We suggest you to run the test and contact us if does not work as expected.
• Q: What is the number of density of cells recommended for ELISA assay (for cell culture supernants and cell lysates)?
  A: The number of cells we recommened for cell culture supernants and cell lysates is 10^6 cells/mL.
• Q: How many samples can I test on one ELISA plate?
  A: On each 96 well plate, we recommend running an 8-point standard curve with duplicate wells. With the remaining 80 wells, 40 samples can be tested in duplicate.
• Q: The volumes of my samples are small, would it be possible to use 50µl/sample?
  A: There are two solutions for insufficient sample volume. <br> 1. Dilute the samples with the provided sample diluent buffer into 100ul. <br> 2. If 50ul of sample is added into a well, add 50ul of standard solution.
• Q: Will this kit work on tissue samples?
  A: For tissue homogenates, endogenous biotin should be considered. Endogenous biotin in tissue homogenates might introduce false positive signal. Please run the test as described below to confirm if there is Endogenous biotin in the tissue lysate samples using the ELISA kit. <br> • Add tissue homogenates into the wells and then add ABC and TMB without adding any biotinylated detection antibody to see if any signal will be observed. <br> • If no signal is produced, then you can work on the tissue sample by using the kit.
• Q: Is it possible to dilute 10X TBS stock that is 250mM Tris Base/Tris-Hcl, 27 mM KCl, and 1.37M NaCl to be used as the wash buffer?
  A: You can dilute the 10X TBS stock to 1XTBS and test the pH value of the 1xTBS. <br> The 1XTBS can be used if the pH value falls in the range of 7.2-7.6.
• Q: Would your kits work normally with samples from Wistar Rat?
  A: Our kits would work normally with samples from Wistar Rat.
• Q: How can I store the dissolved wash buffer for a longer period of time?
  A: You can prepare a concentrated wash buffer (25X) which can be stored at 4°C for 1-3 months.
• Q: Does the TMB color developing agent contain H2O2?
  A: Yes, the TMB color developing agent contain H2O2.
• Q: Does this ELISA kit contain any product produced in humans or primates? <br> Keywords: component, ingredient
  A: None of the components in our ELISA kits are produced in humans or primates.
• Q: What is the volume of the recombinant protein control? What is its shelf life? <br>Keywords: expiration, storage, temperature, size
  A: The volume of the control is around 100pg. If unopened, the shelf life of the control is the same as the whole kit - "Store at 4˚C for 6 months, at -20˚C for 12 months." The reconstituted control can be stored at -20˚C for 2 days.
• Q: Will it be okay to run the experiment if I accidentally used the wrong buffer (e.g. sample diluent buffer) for antibody dilution instead of the antibody diluent buffer? Keywords: dilution, replace, substitute, sample buffer
  A: Using the sample diluent buffer for antibody dilution instead of the antibody diluent buffer will negatively affect the test result to some extent. The values of both standard and sample might be lower than the normal values.
• Q: The protocol says to use neutral PBS and provides a recipe. Could I use neutral PBS made with KCl or KH2PO4 (common constituents for most PBS recipes) instead? Keywords: protocol, alternative buffer, applications, reagent recipe
  A: Yes, it is ok to use common PBS recipes. We have tried many types of buffers with common constituents, and no significant difference was observed whatsoever.
• Q: Why are my O.D. values different than your values on the datasheet? Keyword: troubleshooting, reference
  A: We detected the kit in our lab and got our values on the datasheet before delivery, but protein activity will decrease as time goes on, so you may get lower O.D. values than ours. However, it should still be in a reasonable range and the standards can be used to calculate sample values. Good linearity of curve is more important than the actual numerical value.
• Q: On the ELISA kit datasheet it says "HRP substrate TMB was used to visualize HRP enzymatic reaction." What is HRP and which part of the kit contains HRP? Keyword: kit components, reagents, protocol
  A: It means Avidin-Biotin-Peroxidase Complex(AR1103) and you could find it in Kit Components.
• Q: Do you offer any ELISA kits that can work with whole blood samples instead of plasma or urine? Keyword: applications
  A: We test serum and plasma routinely, and there is very little difference between serum, plasma and whole blood. The whole blood also contains proteins we need to test. The kit can be used to test whole blood in theory.
• Q: Does silicic acid (formula SiOH4) affect the results of the ELISA assay? Keyword: protocol, reagents
  A: The acid may affect the binding of antigen to antibody, it is not recommended to use. That being said, it is unlikely to affect the reaction if the solution remains an overall neutral environment.
• Q: Is there lot-to-lot variation of the ELISA kits? What are Boster's general services when I have questions about your kits? Keyword: technical support, help, customer service
  A: The NIBSC/WHO 1st International Standard is evaluated, we always test our kits before delivery, and customers can find the test result on the protocol. If the customer needs technical support from us to analyze their data, please contact us and we ask that you provide us the following information: the lot# and production date, and when did the customer detect the kit.
• Q: The results of my standards do not look correct, what could be the problem? Keyword: troubleshoot, protocol
  A: Double check the protocol for plate washing. It should be examined on three aspects: 1) Did you make the washing buffer correctly? 2) How long did you let the buffer stay in wells? 3) What was the volume of the washing buffer added to each well? In addition, pay attention when adding sample to avoid contamination. And we suggest testing the standards again. Values of standards at low concentration are more affected than that at higher concentration, so customer can still get expected values at high concentration even if there is an error. If problems persist, please contact technical support with the catalog and lot number of your product.
• Q: What is the well depth of the 96 well plates for the ELISA kits? Keyword: well capacity, product size
  A: The well depth is 300ul, and the max capacity is 350ul. The height of the well is 1.1 cm, and the internal size is 1 cm.
• Q: The kit does not include wash solution, what should I do? Keyword: wash buffer
  A: Our Elisa kits do not come with wash solution, but we have included information about how to make washing buffer on the datasheet. Please refer to the "Material Required But Not Provided" section. We also offer washing buffer for sale separately (Phosphate Buffered Saline Powder SKU: AR0030).
• Q: For your ELISA kits, are the capture and detection antibodies polyclonals or monoclonals? Keyword: antibody clonality
  A: This information can be found for each kit under the "Properties" section, and you can find the immunogen sequence information in the "Overview" section.
• Q: Do your ELISA kits come with sealants or plate covers? Keyword: storage, sequential use
  A: The kit can be used within a month sequentially if it's opened and stored at 4 degree. There is no need to use sealants, the plate can be packaged with aluminum foil bag, and for other reagents keep bottle tightly closed.
• Q: What is the concentration of Sodium Azide in your Elisa kits? Keyword: preservative
  A: Our Elisa kits contain 0.02% Sodium Azide. All of the components except TMB colour developing reagent and stop solution contains 0.02% Sodium azide as the preservative.
• Q: Are there positive and negative controls available for my ELISA kit? Keyword: positive control, negative control
  A: We can provide a recombinant protein as a control. It costs $50 per control and takes 2 weeks to manufacture. We cannot provide a positive or negative control in serum.
• Q: Does the Human APP PicoKine™ ELISA Kit detect all of APP, APP40 and APP42?
  A: The Human APP PicoKine™ ELISA Kit detects extracellular APP with the immunogen sequence L18-L688.
• Q: Why do I get positive results for my knockout (KO) model when used as a control?
  A: The knockout (KO) model may contain truncated forms of the target protein which can be detected by the ELISA assay.
• Q: How long do I soak my plate in the wash buffer?
  A: The plate should soak in 0.3mL PBS or TBS wash buffer for at least 1-2 minutes in an automated wash station.
• Q: Can I use Tween in my wash buffer?
  A: While it is not recommended to use Tween in your wash buffer, small amounts (<0.1% concentration) may decrease background due to insufficient blocking.
• Q: What plate type do I use to set up the microplate reader?
  A: Our plates are made with the Corning costar plates similar to the DNA-BIND 96 -well plates.
• Q: What should I use for negative control?
  A: Please contact us for negative control suggestions. You can also check expression databases such as genecards, uniprot etc. Due to logistic reasons, we do not sell serum or lysates that we use internally for positive or negative control.
• Q: Where can I find troubleshooting information? What should I do if I have unexpected bands, high background, no signal, weak signal
  A: You can find Boster's troubleshoot guides under tech support tab. Please contact us for further assistance on troubleshooting your experiment.
• Q: What is the normal level of this protein in my sample of interest?
  A: We have reviewed literature and have gathered this information for most of our ELISA kits. You can find this information on the product page or contact us if you cannot find it. However this information is only suggestive and cannot replace pilot studies in determining the optimal sample dilution ratio.
• Q: Is the plate separable? Can I use only part of the kit and save the rest for later? How many samples can I run with one kit?
  A: Yes the plate is separable if the kit says that there are removable strips. There are 12 strips of 8 wells each, all removable from the plate. The amount of samples you can run depend on a few factors. In the most common ELISA set up, you will use two strips for standards, and 10 strips for samples using duplicates, which let you run up to 40 samples per kit. Contact us if you have questions regarding other situations.
• Q: Does the kit contain sample preparation reagents? How do I prepare my samples?
  A: Since different sample types require different reagents, we do not include them in the ELISA kit. However we do have each reagent mentioned in the file below available at very reasonable prices. Be sure to check them out. For sample preparation protocols please download the file below: https://www.bosterbio.com/media/pdf/Boster_Sample_Preparation_Protocols.pdf
• Q: Can this ELISA kit work on brain tissue homogenate, cell culture supernatant, saliva, urine, serum, whole blood or any other sample type?
  A: In theory the ELISA kit will work for all sample types if the target protein is present at a level that falls within the linear range of the ELISA kit detection range. We guarantee the kit to work on the sample types that we have tested. If you want dilution ratio suggestions on these sample types please contact our technical support. For sample types that we have not tested for, we suggest you run pilot experiments to decide the optimal sample dilution.
• Q: Can this ELISA kit react with the pro-form of the target protein? Can this ELISA kit react with an isoform of the protein?
  A: In general, unless otherwise specified, the ELISA kit is pan-specific, meaning that it will react with all different forms of the target protein if they share the majority of the target protein's sequence. The capture and detection antibodies are reactive to the entire sequence of the standard protein. You can find the sequence information of the standard protein in the "immunogen" section. For more information about the specificity of the kit for your particular experiment, please contact our technical support.
• Q: Can this ELISA kit react with human, mouse, rat or other species?
  A: If the kit is reactive to another commonly used species (human, mouse, and/or rat), we would have listed it as a separate product. If you want to check cross-reactivity to a species that is not included in the 3 species listed above, please contact our technical support for more information. As a rule of thumb, if the sequences are 90%+ identical, there is a high chance of cross-reactivity for your species of interest.