Elastin Assay – Fastin™ Elastin
The Fastin™ Elastin Assay is a quantitative dye-binding method for the analysis of elastins released into tissue culture medium and extracted from biological materials.
The dye label employed is 5,10,15,20-tetraphenyl-21H,23H-porphine tetra-sulfonate (TPPS).
Elastin forms that can be measured by the Fastin Assay as α-elastin:
- soluble tropoelastins;
- lathyrogenic elastins;
- insoluble elastins (following solubilization to elastin polypeptides [α-elastin, κ-elastin])
The dye reagent binds to the ‘basic’ and ‘non-polar’ amino acid sequences found in mammalian elastins. For analysis of soluble elastin, samples should be free of any particulate material (cell debris, insoluble extracellular matrix material). The presence of other soluble proteins or of complex carboyhdrates does not interfere with the Fastin Assay.
Dye Bound α-Elastin (0, 12.5 and 25μg) after Removal of Unbound Dye from the Tubes.
Q. What is the sensitivity of the Fastin assay?
Q. How long does the Fastin assay take to run?
A. 4 hours
Q. How stable is the Fastin assay?
A. Unopened all the reagents are stable for 1 year at room temperature. Do not freeze kitor remove metal seal from reference standard.
Q. What forms of elastin does the Fastin assay measure?
A. Soluble tropoelastins, lathyrogenic elastins and insoluble cross-linked elastins that have been solubilised using hot oxalic acid treatment.
Q. What absorbance values can samples be measured at?
A. Absorbance peak for TPPS in the dye dissociation reagent is 513nm. However absorbance settings of 545nm or 550nm give suitable readings. A blue green filter is usually appropriate but filters either side of this should be tested to obtain the highest linear absorbance readings possible.
Q. How do I prepare samples for measurement with the Fastin assay?
A. In Vitro Studies – soluble elastin secreted into cell culture medium may be measured directly. Samples should be free from any particulate material. Tropoelastin is the elastin form usually found in cell-culture medium during in vitro cell culture.
A. In Vivo Studies – a method for the extraction of insoluble elastin from tissue and conversion to soluble α-elastin or soluble κ-elastin is given (page 12-13). After solubilisation the samples are assayed by the procedure described for soluble elastin.