H1000 (100 assays)
H2000 (400 assays, MultiPack)
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The kit
Method
The protocol takes tissue samples through a sequential preparation involving enzymatic protein digestion, followed by precipitation and purification of GAGs, culminating in the precipitation of purified hyaluronic acid.
The processed sample is then incubated for 10 minutes with the Purple-Jelley Dye Reagent, forming a coloured product.
The characteristic third absorbance peak is recorded spectrophotometrically at 655nm.
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).
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.
Background
1. Purple-Jelley Dye Reagent (2x 10ml)
2. Hyaluronan Reference Standard (1x 5ml, 0.2mg/ml soluble Hyaluronic Acid)
3. Precipitating Reagent (3x 25ml)
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.
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.
Certain cyanine dyes exhibit a third absorbance peak — the 'J-aggregate' — at around 650nm in the presence of particular biological polymers. Research in the 1960s, notably by Kay et al. (J. Physical Chem. 68, 1896–1906), revealed that proteins, DNA, polar lipids and glycosaminoglycans, including hyaluronic acid, all induce this peak. The Purple-Jelley assay exploits this: purified hyaluronic acid is reacted with the dye reagent, and the resulting absorbance at 655nm is compared against a calibration curve.
In 1936, Edwin Jelley noted in a letter to 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 around 650nm. This extra peak was later termed the 'J-aggregate', in honour of Edwin Jelley. The J-aggregate is now understood to be a supra-molecular complex, formed by stacking of individual dye molecules.
The kit is named for the purple colour of the dye reagent — and for Edwin Jelley himself.
Support
The questions our technical team is asked most about this kit. Not answered here?
Ask us directly0.2μg in 20μl of assayed sample, equivalent to 10μg/ml.
The absorbance peak for Hyaluronic Acid in Purple-Jelley dye reagent is 655nm.
Preparation details for samples in tissue are described in the assay manual.
Removal of tissue protein before assay is essential in order to isolate the HA from potentially dye binding proteins. Sulfated glycosaminoglycans must also be removed before measuring the isolated HA. This is achieved using a two step critical electrolyte salting out process (CEC).
In the literature
Used to measure hyaluronan in tissue biology, especially muscle, fascia and nerve, and in hydrogels and decellularised matrix.
Hyaluronan changes with ageing, stroke and nerve injury.
Hyaluronan in tissue-derived matrices.
Hyaluronan in lymphoma and airway research.
Typical samples
Skeletal muscle & fascia (150–250 mg) · nerve · skin · dECM hydrogels · HA formulations
Often used with
A curated selection of peer-reviewed work using this kit, chosen to span different tissues, models and questions.
Cryobioprinted human tumor models with shelf-stable programmability
Hyaluronan lost from decellularised porcine pancreas. Purple-Jelley was used on native and decellularised porcine pancreas matrix, showing a significant loss of hyaluronan that led the authors to supplement the stroma bioink with hyaluronan in cryopreservable pancreatic tumour–stroma models.
The impact of stroke on intramuscular connective tissue in mice
Hyaluronan in mouse forelimb muscle after stroke. Purple-Jelley was used on about 150 mg of forelimb muscle, digested overnight with proteinase K at 55 °C and read at 650 nm, showing raised hyaluronan on both sides four weeks after cerebral artery occlusion.
Impact of Hyaluronic Acid on the Cutaneous T-Cell Lymphoma Microenvironment: A Novel Anti-Tumor Mechanism of Bexarotene
Hyaluronan in lymphoma-inoculated mouse skin. Purple-Jelley was used to extract and measure hyaluronan at 655 nm in mouse skin injected with T-cell lymphoma cells, comparing vehicle and bexarotene, with the extracted hyaluronan then run on agarose gels for molecular-weight analysis.
Enhancement of lacrimal gland cell function by decellularized lacrimal gland derived hydrogel
Hyaluronan in decellularised lacrimal gland hydrogels. Purple-Jelley was used on 10 mg lyophilised samples of porcine lacrimal gland hydrogel pregels and native tissue digests, showing hyaluronan content rose with longer pepsin hydrolysis, in developing hydrogels for lacrimal gland regeneration in dry eye.
Unraveling the Relevance of Tissue-Specific Decellularized Extracellular Matrix Hydrogels for Vocal Fold Regenerative Biomaterials: A Comprehensive Proteomic and In Vitro Study
Hyaluronan in vocal-fold matrix hydrogels. Purple-Jelley was used on native porcine vocal fold, decellularised vocal-fold and intestinal matrices, and fibroblast-laden hydrogels cultured for up to 14 days, measuring hyaluronan lost on decellularisation and newly deposited by the cells.
The Effects of Aging on the Intramuscular Connective Tissue
Hyaluronan in young and aged human and mouse muscle. Purple-Jelley was used on about 250 mg of human quadriceps and mouse hindlimb muscle, digested overnight with proteinase K at 55 °C and read at 655 nm, to compare hyaluronan content across age groups.
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