Tissues such as lungs, heart and blood vessels need to be able to stretch and recoil repeatedly throughout an organism's life. The protein Elastin is largely responsible for this elasticity. Elastin is initially secreted by elastin-producing cells as a soluble monomeric precursor known as tropoelastin. This undergoes a process of self-assembly and cross-linking with other extracellular matrix components that results in the formation of 'insoluble' elastin fibres.
The Biocolor Fastin assay is a user-friendly, dye-based means of quantifying elastins derived from both in-vivo and in-vitro sources. A variety of elastin forms can be assayed, from the immature tropoelastin to mature ‘insoluble’ elastin fibres.
Further information on how the assay works can be found on the 'Mode of Action' tab.
A list of suggested sample types can be found under the 'Assay Specification' tab.
The Fastin Dye Reagent contains an elastin-binding synthetic porphyrin, TPPS (5,10,15,20-tetraphenyl- 21H,23H-porphine). This affinity of TPPS for elastin was first observed when used as a 'vital stain' on live animals. Most tissues took up the dye initially but only elastin retained the TPPS molecules over time. [Winkelman, J. (1962), Cancer Res. 22, 589-596; Winkelman, J & Spicer, S. (1962), Stain Technol. 37, 303-305].
It has been proposed that the elastin binding of TPPS may be due to the retention of the acidic dye (which contains four charged sulfate groups) by the basic amino acid side chain residues of elastin.
Step 1. Incubation of samples containing soluble or solubilised elastin with the Fastin Dye Reagent causes an elastin-dye complex to form. This insoluble complex then precipiates.
Step 2. Dye-labelled elastin is then isolated by centrifugation and the unbound dye removed. Elastin-bound dye is then eluted and measured spectrophotometrically.
Step 3. The elastin content of unknown samples can be calculated by comparison against a calibration curve prepared using a standard comprising water-soluble elastin (supplied with the kit).
In-vivo: tissues and fluids. Insoluble elastin will first require conversion to water soluble α-elastin using the oxalic acid reagents and extraction protocol supplied with the kit.
In-vitro: Elastin produced by cells during 2D/3D cell culture. NB elastin in conditioned cell media is typically below the detection limit of the kit.
This kit is designed for research use only. Not for use in diagnostic procedures.
Kit requires access to a centrifuge, heated water bath or block, as well as a spectrophotometer or colorimeter capable of absorbance detection at 513nm.
Specific sample preparation protocols may require customer to provide further reagents, consult assay manual for further information.
1. Dye Reagent (1x110ml)
2. α-elastin Reference Standard (1x5ml, 1.0 mg/ml soluble Bovine elastin)
3. Oxalic Acid (1x20ml) Precipitating Reagent (1x30ml)
4. Dye Dissociation Reagent (1x30ml)
5. Precipitating Reagent (1x30ml)
6. 1.5ml micro-centrifuge tubes for dye-labelling reaction.
7. 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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