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Human VEGF DuoSet ELISA

R&D Systems | Catalog # DY293B

R&D Systems
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Key Product Details

Assay Type

Solid Phase Sandwich ELISA

Assay Range

31.2-2000 pg/mL

Sample Type

Cell culture supernates, serum, and plasma
Note: Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet

Reactivity

Human

Human VEGF DuoSet ELISA 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
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Product Summary for Human VEGF DuoSet ELISA

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human VEGF. 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 Specifications

Assay Format

96-well strip plate (sold separately)

Sample Volume Required

100 µL

Detection Method

Colorimetric ELISA - 450nm (TMB)

Conjugate

Biotin

Label

HRP

Scientific Data Images for Human VEGF DuoSet ELISA

Human VEGF ELISA Standard Curve

Human VEGF ELISA Standard Curve

Human VEGF DuoSet ELISA

Human VEGF DuoSet ELISA

Effects of cell stress on VEGF-A and HIF1 alpha expression in Müller cells and Y79 photoreceptors. (A) VEGF-A was measured by ELISA using conditioned media collected from MIO-M1 and Y79 cells, n = 4–5/group in Müller cells and n = 8/group in Y79 photoreceptors, ** p < 0.01; (B) Western blots for HIF1 alpha using cellular proteins, n = 4/group, ** p < 0.01. For MIO-M1 Müller cells: Grey bars: 5 mM glucose. Black bars: 25 mM glucose. For Y79 photoreceptors: Grey bars: 11 mM glucose. Black bars: 25 mM glucose. NS, not significant; LG, low glucose; HG, high glucose. Image collected and cropped by CiteAb from the following open publication (https://www.mdpi.com/1422-0067/18/3/533), licensed under a CC-BY license. Not internally tested by R&D Systems.

Kit Contents for Human VEGF DuoSet ELISA

  • 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)

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: VEGF

Vascular endothelial growth factor (VEGF or VEGF-A), also known as vascular permeability factor (VPF), is a potent mediator of both angiogenesis and vasculogenesis in the fetus and adult (1-3). It is a member of the PDGF family that is characterized by the presence of eight conserved cysteine residues in a cystine knot structure and the formation of antiparallel disulfide-linked dimers (4). Humans express alternately spliced isoforms of 121, 145, 165, 183, 189, and 206 amino acids (aa) in length (4). VEGF165 appears to be the most abundant and potent isoform, followed by VEGF121 and VEGF189 (3, 4). Isoforms other than VEGF121 contain basic heparin-binding regions and are not freely diffusible (4). Human VEGF165 shares 88% aa sequence identity with corresponding regions of mouse and rat VEGF. VEGF is expressed in multiple cells and tissues including skeletal and cardiac muscle (5, 6), hepatocytes (7), osteoblasts (8), neutrophils (9), macrophages (10), keratinocytes (11), brown adipose tissue (12), CD34+ stem cells (13), endothelial cells (14), fibroblasts, and vascular smooth muscle cells (15). VEGF expression is induced by hypoxia and cytokines such as IL-1, IL-6, IL-8, oncostatin M, and TNF-alpha (3, 4, 9, 16). VEGF isoforms are differentially expressed during development and in the adult (3). 
VEGF dimers bind to two related receptor tyrosine kinases, VEGF R1 (also called Flt-1) and VEGF R2 (Flk-1/KDR), and induce their homodimerization and autophosphorylation (3, 4, 7, 17, 18). These receptors have seven extracellular immunoglobulin-like domains and an intracellular split tyrosine kinase domain. They are expressed on vascular endothelial cells and a range of non-endothelial cells. Although VEGF affinity is highest for binding to VEGF R1, VEGF R2 appears to be the primary mediator of VEGF angiogenic activity (3, 4). VEGF165 also binds the semaphorin receptor, neuropilin-1, which promotes complex formation with VEGF R2 (19). 
VEGF is best known for its role in vasculogenesis. During embryogenesis, VEGF regulates the proliferation, migration, and survival of endothelial cells (3, 4), thus regulating blood vessel density and size, but playing no role in determining vascular patterns. VEGF promotes bone formation through osteoblast and chondroblast recruitment and is also a monocyte chemoattractant (20-22). After birth, VEGF maintains endothelial cell integrity and is a potent mitogen for micro- and macro-vascular endothelial cells. In adults, VEGF functions mainly in wound healing and the female reproductive cycle (3). In diseased tissues, VEGF promotes vascular permeability. It is thus thought to contribute to tumor metastasis by promoting both extravasation and tumor angiogenesis (23, 24). Various strategies have been employed therapeutically to antagonize VEGF-mediated tumor angiogenesis (25). Circulating VEGF levels correlate with disease activity in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus (26).

Long Name

Vascular Endothelial Growth Factor

Alternate Names

MVCD1, VAS, Vasculotropin, VEGF-A, VEGFA, VPF

Entrez Gene IDs

7422 (Human); 22339 (Mouse); 83785 (Rat); 281572 (Bovine); 403802 (Canine); 493845 (Feline); 30682 (Zebrafish)

Gene Symbol

VEGFA

Additional VEGF Products

Product Documents for Human VEGF DuoSet ELISA

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Human VEGF DuoSet ELISA

For research use only

Citations for Human VEGF DuoSet ELISA

Customer Reviews for Human VEGF DuoSet ELISA (14)

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Showing  1 - 5 of 14 reviews Showing All
Filter By:
  • Human VEGF DuoSet ELISA
    Name: Anonymous
    Sample Tested: Supernatant from cultured human dermal fibroblasts (HDFa) and human dermal fibroblast
    Verified Customer | Posted 06/28/2023
    Human VEGF DuoSet ELISA standard curve
    Human VEGF DuoSet ELISA DY293B
  • Human VEGF DuoSet ELISA
    Name: Gabriele Strusi
    Sample Tested: Human cell conditioned medium and HepG2 human hepatocellular carcinoma cell line
    Verified Customer | Posted 11/21/2022
    Human VEGF DuoSet ELISA DY293B
  • Human VEGF DuoSet ELISA
    Name: Anonymous
    Sample Tested: Cell culture supernatant
    Verified Customer | Posted 05/12/2021
    Human VEGF DuoSet ELISA DY293B
  • Human VEGF DuoSet ELISA
    Name: Carmen Martin-Ruiz
    Sample Tested: Human brain tissue homogeneate and Brain tissue
    Verified Customer | Posted 08/14/2019
    Straight forward protocol, used on human tissue homogeneates on a 384-well format with 450/550 correction.
    Human VEGF DuoSet ELISA DY293B
  • Human VEGF DuoSet ELISA
    Name: Amnon Berger
    Sample Tested: Cell culture supernatant
    Verified Customer | Posted 03/23/2019
    I use this kit to measure VEGFA levels in cell culture supernatant with very clear results. The standard curve is great and redundant.
    Human VEGF DuoSet ELISA DY293B
  • Human VEGF DuoSet ELISA
    Name: Sophie Millar
    Sample Tested: Cell culture supernatant and Cell Culture Media
    Verified Customer | Posted 01/16/2019
    Human VEGF DuoSet ELISA DY293B
  • Human VEGF DuoSet ELISA
    Name: Anonymous
    Sample Tested: Cell culture supernatant
    Verified Customer | Posted 10/11/2018
  • Human VEGF DuoSet ELISA
    Name: Anonymous
    Sample Tested: Cell culture medium samples and medium samples
    Verified Customer | Posted 06/07/2018
    Great kit, very easy to follow!
    Human VEGF DuoSet ELISA DY293B
  • Name: Anonymous
    Sample Tested: THP-1 human acute monocytic leukemia cell line
    Verified Customer | Posted 07/17/2017
  • Human VEGF DuoSet ELISA
    Name: Anonymous
    Sample Tested: A549 human lung carcinoma cell line
    Verified Customer | Posted 06/23/2017
    Human VEGF DuoSet ELISA DY293B
  • Human VEGF DuoSet ELISA
    Name: Abby Sukarto
    Sample Tested: Human tissue
    Verified Customer | Posted 05/03/2017
    The substrate incubation resulted in weak change of color and so longer incubation is required for human VEGF ELISA kit.
  • Human VEGF DuoSet ELISA
    Name: Anonymous
    Sample Tested: Various cell lines
    Verified Customer | Posted 03/14/2017
    VEGF quantification was performed onto various cell lines and data obtained is presented with the chart attached
    Human VEGF DuoSet ELISA DY293B
  • Human VEGF DuoSet ELISA
    Name: Ricardo Rivera
    Sample Tested: Cell culture supernatant
    Verified Customer | Posted 08/13/2016
    Human VEGF DuoSet ELISA DY293B
  • Human VEGF DuoSet ELISA
    Name: Anonymous
    Sample Tested: Various cell lines and Various cell lines were cultured and supernatant obtained to study time course VEGF secretion
    Verified Customer | Posted 05/23/2016
    Human VEGF DuoSet ELISA DY293B

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Protocols

View specific protocols for Human VEGF DuoSet ELISA (DY293B):

GENERAL ELISA PROTOCOL

Plate Preparation

  1. 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.
  2. 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.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. 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.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. 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.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. 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.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. 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.

 

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FAQs for Human VEGF DuoSet ELISA

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  • Q: What forms of VEGF does Human VEGF DuoSet ELISA (Catalog # DY293B) detect?

    A: The Human VEGF DuoSet ELISA, Catalog # DY293B, has been shown to recognize rhVEGF165, rhVEGF121, and rhVEGF165b. Additional information regarding our specificity testing can be found in the product datasheet. 

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