Hyaluronic acid, in its hydrated form, is a unique polysaccharide polymer, sometimes referred to as a 'gentle giant'! It is found in many tissues, but particularly in cartilage and synovial fluid and consists of a lengthy, flexible, non-branching chain with a repeating disaccharide pattern. This pattern comprises alternating uronic acid and aminosugar units. Hyaluronic acid is involved in a wide range of roles including tissue hydration and lubrication, cell signalling, extracellular matrix reorganisation and wound repair.
This involvement of Hyaluronic acid in tissue lubrication and wound healing has meant it has become a well-known additive to both medical and cosmetic skincare treatments, as well as treatments for osteoarthritis. The ability to assay or measure hyaluronic acid is therefore of interest to scientific researchers from a range of disciplines.
Discovering the J-Aggregate Effect in Cyanine Dyes
In 1936, Edwin Jelley noted in a letter to the journal Nature(Nature, 138, 1009 - 1010) that when certain cyanine dyes were dissolved in deionized water, they displayed a double peak at approximately 540nm and 570nm, however when dissolved in 5 M NaCl, they exhibited a third absorbance peak at a around 650nm. This extra peak was later termed the 'J-aggregate,' in honour of Edwin Jelley. This J-aggregate is now understood to be a supra-molecularcomplex, formed by stacking of individual dye molecules.
Subsequent research in the1960s, notably by Kay et al. (J. Physical Chem. 68, 1896 – 1906), revealed that various biological polymers, such as proteins, DNA, polar lipids, and glycosaminoglycans (including Hyaluronic acid), also induce this third absorbance peak. This phenomenon is exploited in the ‘Purple-Jelley’ assay, named after the purple colour of the dye reagent and Edwin Jelley himself!
During the assay, hyaluronic acid is selectively purified via assay's sample preparation protocol. This is then reacted with the Purple-Jelley dye reagent, 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).
A list of suggested sample types can be found under the 'Assay Specification' tab.
In-vivo: Hyaluronic acid purified from in-vivo tissues. The kit protocol involves extraction and purification of hyaluronic acid prior to reaction with the Purple-Dye reagent.
In-vitro:Measurement of hyaluronic acid from in-vitro cellular material is possible, but may require adaption of the protocol. Please contact us for further information.
This kit is designed for research use only. Not for use in diagnostic procedures.
Kit requires access to a centrifuge, as well as a spectrophotometer/colorimeter capable of colorimetric, absorbance detection at 655nm.
Specific sample preparation protocols may require customer to provide further reagents, consult assay manual for further information.
1. Purple-Jelley Dye Reagent (1x 20ml)
2. Hyaluronan Reference Standard (1x 5ml, 0.2mg/ml soluble Hyaluronic Acid)
3. Precipitating Reagent (2x 34ml)
4. Sodium Chloride (1x 20ml)
5. Cetylpyridinium Chloride (1x 20ml)
6. TRIS-buffered Saline (5x tablets)
7. 2ml screw-cap tubes for preparation of samples.
8. Assay kit manual
NB: Additional reagents may be required for sample preparation prior to assay. Consult manual or contact us for further details.
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