Description
The Biocolor Purple-Jelley Hyaluronic Acid Assay is a quantitative, dye-based method for measurement of hyaluronic acid/hyaluronate/hyaluronan derived from tissues.
The assay manual includes methods for the user to remove tissue protein and sulfated glycosaminoglycans before measuring the isolated hyaluronic acid (HA).
Principle of Method
During the assay, hyaluronic acid is selectively purified during the assay sample preparation protocol. This is then reacted with the Purple-Jelley dye reagent (containing ‘Stains-all’ dye), and the absorption of the characteristic third wavelength recorded. By comparison with a calibration curve the hyaluronic acid content of the sample can be measured.
Step 1. The assay protocol takes tissue samples through a sequential sample preparation protocol which involves enzymatic protein digestion, followed by precipitation and purification of GAGs, culminating in the precipitation of purified Hyaluronic acid.
Step2. The processed sample is then incubated for 10 minutes with the Purple-Jelley dye reagent, forming a coloured product which can be measured spectrophotometrically.
Step 3. The Hyaluronic acid content of unknown samples can be calculated by comparison against a calibration curve prepared using a standard comprising hyaluronic acid (supplied with the kit).
Sample Type
Tissue extracts.
Removal of tissue protein before assay is essential in order to isolate the HA from potentially dye binding proteins; and sulfated glycosaminoglycans need to be removed before measuring the isolated HA, from a two step critical electrolyte salting out process (CEC). Methods for protein removal and sGAG removal can be found in the product manual.
Specifications
| Detection Method | Colorimetric Detection (655 nm) |
| Assay Range | 0 - 100 µg/ml |
| Limit of Detection | 10 µg/ml |
| Sensitivity | 0.2 µg |
| Measurements Per Kit | 100 in total (allows a maximum of 46 samples to be run in duplicate alongside a standard curve) |
Kit Contents
The standard size Purple-Jelley™ Hyaluronic Acid kit contains:
- Purple-Jelley Dye Reagent (1x 20 ml)
- Hyaluronan Reference Standard (1x 5 ml, 0.2 mg/ml soluble Hyaluronic Acid)
- Precipitating Reagent (2x 34 ml)
- Sodium Chloride (1x 20 ml)
- Cetylpyridinium Chloride (1x 20 ml)
- TRIS-buffered Saline (5x tablets)
- 2 ml screw-cap tubes for preparation of samples
- Assay kit manual
Examples of Results
The distribution of HA in tissues as produced in this assay procedure is listed in Table 1 and is given only as an outline guide. In adult animals the HA percentage gradually increases during ageing as muscle and fat mass decreases.
Other Names/Synonyms
Hyaluronic acid, Hyaluronan, HA, Purple-Jelly Hyaluronan Assay
Resources
- Product Datasheet (PDS)
- Product References
- Material Safety Data Sheet (MSDS)
- Frequently Asked Questions (FAQ)
Related Products
- Extracellular Matrix Assays
- Proteinase K
- 3-D Life PVA-HA Hydrogel Kit
- 3-D Life Dextran-HA Hydrogel Kit
-
Hyaluronidase Enzyme (Bovine purified)
About Hyaluronic Acid
Hyaluronic acid (HA), also known as hyaluronan, is a non-sulphated glycosaminoglycan (GAG) and is composed of repeating polymeric disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine linked by a glucuronidic β (1→3) bond. In aqueous solutions HA forms specific stable tertiary structures. Despite the simplicity in its composition, without variations in its sugar composition or without branching points, HA has a variety of physicochemical properties. HA polymers occur in a vast number of configurations and shapes, depending on their size, salt concentration, pH, and associated cations. Unlike other GAG, HA is not covalently attached to a protein core, but it may form aggregates with proteoglycans. HA encompasses a large volume of water giving solutions high viscosity, even at low concentrations.
HA is widely distributed, from prokaryotic, to eukaryotic cells. In humans, HA is most abundant in the skin, (accounting for 50% of the total body HA), the vitreous of the eye, the umbilical cord, and synovial fluid, but it is also present in all tissues and fluids of the body, such as skeletal tissues, heart valves, the lung, the aorta, the prostate, tunica albuginea, corpora cavernosa and corpus spongiosum of the penis. HA is produced primarily by mesenchymal cells but also by other cell types.
Considerable evidence exists regarding the functional role of HA in molecular mechanisms and, consequently, the potential role of HA for the development of novel therapeutic strategies for many diseases.
Functions of HA include the following: hydration, lubrication of joints, a space filling capacity, and the framework through which cells migrate. The synthesis of HA increases during tissue injury and wound healing and HA regulates several aspects of tissue repair, including activation of inflammatory cells to enhance immune response and the response to injury of fibroblasts and epithelial cells. HA also provides the framework for blood vessel formation and fibroblast migration, that may be involved in tumor progression. The correlation of HA levels on the cell surface of cancer cells with the aggressiveness of tumors has also been reported.
The size of HA appears to be of critical importance for its various functions described above. HA of high molecular size, usually in excess of 1,000 kDa, is present in intact tissues and is antiangiogenic and immunosuppressive, whereas smaller polymers of HA are distress signals and potent inducers of inflammation and angiogenesis.
Want to learn more about Hyaluronan? We have compiled the links below which contain information that you may find interesting.
- Hyaluronic acid as a bioactive component for bone tissue regeneration: Fabrication, modification, properties, and biological functions
- The application of hyaluronic acid in bone regeneration
- Hyaluronic Acid-Based Hydrogels: from a Natural Polysaccharide to Complex Networks
Ilex Life Sciences LLC is an official distributor of Biocolor products.