Human Aggrecan DuoSet ELISA

Catalog # Availability Size / Price Qty
DY1220
Ancillary Products Available
Human Aggrecan ELISA Standard Curve
1 Image
Product Details
Procedure
Citations (3)
FAQs
Supplemental Products
Reviews

Human Aggrecan DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
100 µL
Sufficient Materials
For fifteen 96-well plates*
Specificity
Please see the product datasheet

* 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 Aggrecan. 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 ELISA.

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


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 equivalent

Reagent Diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).

Blocking Buffer: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).

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), or equivalent

Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent

 

Data Example

Human Aggrecan ELISA Standard Curve

Product Datasheets

Preparation and Storage

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

Background: Aggrecan

Aggrecan belongs to the chondroitin sulfate (CS) proteoglycan family, which also includes Versican, Brevican, and Neurocan. Each Aggrecan molecule contains approximately 100 and 30 keratan sulfate and glycosaminoglycan (GAG) sidechains, respectively. Aggrecan non-covalently associates with hyaluronan via the link modules and an Ig domain in its N-terminus. It is the most abundant proteoglycan in cartilage, and contributes to the load-bearing capacity of this tissue.

Entrez Gene IDs:
176 (Human)
Alternate Names:
ACAN; AGC1; AGC1SEDK; aggrecan core protein; Aggrecan; Cartilage-specific proteoglycan core protein; Chondroitin sulfate proteoglycan 1; Chondroitin sulfate proteoglycan core protein 1; CSPCP; CSPG1; CSPG1aggrecan 1; CSPGCP; large aggregating proteoglycan; MSK16; MSK16AGCAN; SEDK

Assay Procedure

GENERAL ELISA PROTOCOL

Plate Preparation

  1. Dilute the Capture Antibody (to the working concentration stated in the product datasheet ) 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 each well of the microplate as recommended in the product datasheet. Incubate at room temperature for a minimum of 1 hour.

    Note: The recommended Reagent Diluent typically contains 1% BSA. Some DuoSet Development Kits require alternative blocking agents, or for plates to be blocked overnight with a higher percentage of BSA, please see the product datasheet for details.
     
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

 

PRECAUTION
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.

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 (as recommended in the product datasheet), 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.

Citations for Human Aggrecan 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.

3 Citations: Showing 1 - 3
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  1. Sialoglycoprotein isolated from eggs of Carassius auratus promotes fracture healing in osteoporotic mice
    Authors: F Wang, L Han, X Wang, Y Li, Y Zhu, J Wang, C Xue
    J Food Drug Anal, 2018;26(2):716-724.
    Species: Mouse
    Sample Types: Serum
  2. Identification of Novel Chondroprotective Mediators in Resolving Inflammatory Exudates
    Authors: MK Kaneva, KV Greco, SE Headland, T Montero-Me, P Mori, K Greenslade, C Pitzalis, A Moore, M Perretti
    J. Immunol, 2017;0(0):.
    Species: Human
    Sample Types: Cell Culture Supernates
  3. Knockout of Apolipoprotein E in rabbit promotes premature intervertebral disc degeneration: A new in vivo model for therapeutic approaches of spinal disc disorders
    Authors: A Beierfu beta, H Dietrich, C Kremser, M Hunjadi, A Ritsch, T Rülicke, C Thomé, DS Mern
    PLoS ONE, 2017;12(11):e0187564.
    Species: Rabbit
    Sample Types: Tissue Homogenates

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