Human FAP DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Fibroblast Activation Proteinalpha/FAP. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: Fibroblast Activation Protein alpha/FAP
FAP (fibroblast activation protein-alpha; also known as seprase) is a type II transmembrane serine protease that degrades gelatin, Type I collagen, and N-terminal prolines. FAP is induced on reactive stromal fibroblasts in epithelial cancers, sarcomas, and granulation tissue, and may play roles in tumor invasion, tissue remodeling, and wound repair.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Human FAP DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 9
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Colorectal cancer cell intrinsic fibroblast activation protein alpha binds to Enolase1 and activates NF-&kappaB pathway to promote metastasis
Authors: Z Yuan, H Hu, Y Zhu, W Zhang, Q Fang, T Qiao, T Ma, M Wang, R Huang, Q Tang, F Gao, C Zou, X Gao, G Wang, X Wang
Cell Death & Disease, 2021-05-25;12(6):543.
Species: Human
Sample Types: Cell Culture Supernates
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Effect of &alpha2-plasmin inhibitor heterogeneity on the risk of venous thromboembolism
Authors: B Baráth, R Bogáti, T Miklós, J Kállai, ZA Mezei, Z Bereczky, L Muszbek, É Katona
Thrombosis Research, 2021-05-11;203(0):110-116.
Species: Human
Sample Types: Plasma
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Incorporation of &alpha2-Plasmin Inhibitor into Fibrin Clots and Its Association with the Clinical Outcome of Acute Ischemic Stroke Patients
Authors: Z Bagoly, B Baráth, R Orbán-Kálm, I Szegedi, R Bogáti, F Sarkady, L Csiba, É Katona
Biomolecules, 2021-02-25;11(3):.
Species: Human
Sample Types: Plasma
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Regulation of Fibroblast Activation Protein by Transforming Growth Factor Beta-1 in Glioblastoma Microenvironment
Authors: E Krepela, Z Vanickova, P Hrabal, M Zubal, B Chmielova, E Balaziova, P Vymola, I Matrasova, P Busek, A Sedo
International Journal of Molecular Sciences, 2021-01-21;22(3):.
Species: Human
Sample Types: Cell Lysates
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Altered Tissue and Plasma Levels of Fibroblast Activation Protein-&alpha (FAP) in Renal Tumours
Authors: JD Solano-Itu, P Errarte, MC Etxezarrag, E Echevarria, J Angulo, JI López, G Larrinaga
Cancers (Basel), 2020-11-16;12(11):.
Species: Human
Sample Types: Plasma
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Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen
Authors: J Liu, Z Zuo, M Zou, T Finkel, S Liu
PLoS Genet, 2020-10-15;16(10):e1009068.
Species: Human
Sample Types: Cell Fragments
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Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP)
Authors: TW Bainbridge, DR Dunshee, NM Kljavin, NJ Skelton, J Sonoda, JA Ernst
Sci Rep, 2017-10-02;7(1):12524.
Species: Human
Sample Types: Serum
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Circulating fibroblast activation protein activity and antigen levels correlate strongly when measured in liver disease and coronary heart disease
Authors: S Uitte de W, FM Keane, DG Bowen, JJMC Malfliet, HE Zhang, B Maneck, GW McCaughan, FWG Leebeek, DC Rijken, MD Gorrell
PLoS ONE, 2017-06-05;12(6):e0178987.
Species: Human
Sample Types: Plasma
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Heterogeneity of molecular forms of dipeptidyl peptidase-IV and fibroblast activation protein in human glioblastomas.
Authors: Matrasova I, Busek P, Balaziova E, Sedo A
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2017-04-26;161(3):252-260.
Species: Human
Sample Types: Tissue Homogenates
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Human Plasma samples diluted between 1/100 and 1/200 fit perfectly in the standard curve.
we used this kit for quantifying human FAP in serum samples. This kit works well and detects FAP very efficiently in human serum.
