Biocolor’s Purple-Jelley assay kit is the perfect tool for accurate measurement of hyaluronic acid / Hyaluronan levels in your samples. This colorimetric assay is optimised for quantitative analysis in-vivo, tissue-derived hyaluronic acid / Hyaluronan and includes full step-by-step instructions.
Colorimetric Detection (655nm) (Endpoint)
Hyaluronic Acid: A gentle giant!
Hyaluronic acid, in its hydrated form, is a unique carbohydrate polymer, often referred to as a ‘gentle giant.’ It consists of a lengthy, flexible, non-branching chain with a repeating disaccharide pattern. This disaccharide is composed of alternating uronic acid and aminosugar units.
Why is our kit called ‘Purple-Jelley’?
Discovering the J-Aggregate Effect in Cyanine DyesIn 1936, Edwin Jelley made a fascinating observation, documented it in a letter to Nature (Nature 138, 1009 – 1010). He noted a peculiar behaviour of certain cyanine dyes, that when dissolved in 5 M NaCl, they dyes exhibited a third absorbance peak at a longer wavelength, around 650nm. In deionized water, however, they displayed only a double peak at approximately 540nm and 570nm. The 650nm peak in concentrated dye solutions resulted from the aggregation of dye molecules and was later termed a ‘J-aggregate,’ in honor of Edwin Jelley. The J-aggregate is known as a supra-molecular complex, formed by stacking individual dye molecules.
Subsequent research in the 1960s, notably by Kay et al. (J. Physical Chem. 68, 1896 – 1906), revealed that various biological polymers, including proteins, DNA, polar lipids, and glycosaminoglycans, could also induce this third absorbance peak. This phenomenon led to the development of the Purple-Jelley assay, named after the purple color of the dye reagent and Edwin Jelley himself.
An overview of the Purple-Jelley assay steps:
During the assay, hyaluronic acid is selectively purified during the assay 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).
Assay range
10 – 100µg/ml
Limit of Detection
10µg/ml
Detection Method
Colorimetric Detection (655nm) (Endpoint)
Measurements per kit
100 in total (allows a maximum of 46 samples to be run in duplicate alongside a standard curve).
Suitable Samples
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.
Precautions
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.
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.
Document
Biocolor’s Purple-Jelley assay kit is the perfect tool for accurate measurement of hyaluronic acid / Hyaluronan levels in your samples. This colorimetric assay is optimised for quantitative analysis in-vivo, tissue-derived hyaluronic acid / Hyaluronan and includes full step-by-step instructions.
Cell Culture Plate provide excellent smoothness and uniformity based on accurate molding technology. By such property, clear view can be available when examine with a microscope.
Specifications: 6wells,
12wells, 24wells., 48wells, 96wells
PRODUCT FEATURES
The product is made of medical grade USP CLASS VI polymer polystyrene
The product is made under a 100,00- class dust-free manufacturing site
Two kinds of product line up are providing.
For adherent cell culture: Initial adherence and proliferative property of cells via hydrophilic surface treatment.
For suspension cell culture: The surface is resistant to cell adherence, which minimizes damage or loss of cell.
Gamma radiation sterilization
Non-Pyrogenic, DNase/Rnase free.
Document
Cell Culture Plate provide excellent smoothness and uniformity based on accurate molding technology. By such property, clear view can be available when examine with a microscope.
Buffer system mainly for stabilizing protein/enzyme and performance
B buffer
0.15ml
1 Tube
Mainly activated systems such as magnesium ions
Positive control template
0.1ml
1 Tube
Mainly the positive plasmid template is used to test the effectiveness of the kit
Positive control primer mix
0.06ml
1 Tube
Mainly the primer combination of the positive control template
Reagent Guide Manua
16T/bags,48T/Box
3 bags
Reagent technology of protein/enzyme system: freeze-dried powder, freeze-dried microspheres
Principle overview
This kit is based on a room temperature and constant temperature nucleic acid rapid amplification technology: at room temperature and constant temperature, the recombinase and primer form the protein/single-stranded nucleotide complex Rec/ssDNA, with the help of auxiliary proteins and single-stranded binding protein SSB , invade the double-stranded DNA template; form a D-loop region at the invasion site, and start scanning the DNA double-strands; after finding the target region complementary to the primer, the Rec/ssDNA complex disintegrates, and the polymerase also binds to The 3′ end of the primer initiates chain extension. This kit relies on the action of exonuclease at 39 ºC, adding specific molecular probes designed based on the template, and using fluorescence monitoring equipment to achieve real-time monitoring of the amplification process of the target fragment.
Primer design
It is recommended to use primers with a length of 30-35 bp. Primers that are too short will affect amplification speed and detection sensitivity; primers are designed to avoid the formation of secondary structures that affect amplification; the amplicon length is recommended to be 150-300 bp, usually no more than 500 bp.
Fluorescent probe design
The probe sequence does not overlap with the specific primer recognition site, is 46-52 nt in length, and the sequence avoids palindromic sequences, internal secondary structures, and continuous repeated bases. The probe has four modification sites: the middle position ≥ 35 nt from the 5′ end is labeled with a dSpacer (tetrahydrofuran, THF) as the recognition site for exonuclease; the upstream of the THF site is labeled with a fluorescent group, and the downstream Label a quenching group, the distance between the two groups is 2-4 nt; THF is ≥15 nt from the 3′ end, and the 3′ end is labeled with a modifying group, such as an amine group, a phosphate group or a C3-Spacer.
Product features and advantages:
This kit has the advantages of high sensitivity, strong specificity,and short reaction time (only 20 minutes), and the reaction groups are in dry powder state, which is easy to operate and easy to store.
It can be applied to various brands of fluorescence quantitative PCR instruments, constant temperature fluorescence amplification instruments and other fluorescence detection equipment.
Document
This kit is based on a room temperature and constant temperature nucleic acid rapid amplification technology: at room temperature and constant temperature, the recombinase and primer form the protein/single-stranded nucleotide complex Rec/ssDNA, with the help of auxiliary proteins and single-stranded binding protein SSB , invade the double-stranded DNA template; form a D-loop region at the invasion site, and start scanning the DNA double-strands; after finding the target region complementary to the primer, the Rec/ssDNA complex disintegrates, and the polymerase also binds to The 3′ end of the primer initiates chain extension. This kit relies on the action of exonuclease at 39 ºC, adding specific molecular probes designed based on the template, and using fluorescence monitoring equipment to achieve real-time monitoring of the amplification process of the target fragment.
