Biocolor Fastin™ Elastin Assay - Frequently Asked Questions (FAQ)

 

Here you will find answers to many of the frequently asked questions about Biocolor Fastin™ Elastin Assay.

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What is the Fastin Elastin Assay?

The Biocolor Fastin™ Elastin Assay is a quantitative dye-binding method for the analysis of elastins released into tissue culture medium and extracted from biological materials.

General Questions

What forms of elastin does the Fastin Elastin Assay measure?

Soluble tropoelastins, lathyrogenic elastins and insoluble cross-linked elastins that have been solubilized using hot oxalic acid treatment.

What absorbance values can samples be measured at?

Absorbance peak for TPPS in the dye dissociation reagent is 513 nm. However, absorbance settings of 545 nm or 550 nm also give usable readings. A blue green filter is usually appropriate but filters either side of this should tested to obtain the highest linear absorbance readings possible.

How long does Fastin Elastin Assay take to run?

4 hours (following sample extraction).

How stable is the Fastin Elastin Assay?

Unopened, all the reagents are stable at room temperature for 1 year from the date of manufacture. After opening, follow storage conditions indicated on each component label. Do not freeze the kit or remove the metal seal from the reference standard until ready to use.

Sample Processing

How do I prepare samples for measurement using the Fastin Elastin Assay?

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 culture.

In-Vivo Studies: a method for the extraction of insoluble elastin from tissue and conversion to soluble α-elastin or κ-elastin is given (see page 4 of the assay manual). After solubilization, the samples are assayed by the procedure described for soluble elastin.

My sample appears to be undigested following oxalic acid extraction, what can I do?

 The product manual suggests 60 minute extractions; this balances efficiency with convenience. It is possible to extend the extraction time beyond 60 minutes without issue. You could try extending each extraction to 3 hours or even overnight. Please note that multiple extractions may be required (see page 4 of the manual for details).

The efficiency of the elastin extraction can be increased by increasing the sample surface area. If sufficient tissue is available this can be achieved by grinding or processing the samples to a powder prior to addition of the oxalic acid. Practically, this can be aided by lyophilization or snap freezing and grinding the material in the frozen state. Such material should extract more quickly.

Assay Methodology

Why are my standards or samples stained green (rather than red/brown)?

The Fastin dye/elastin labelling interaction is most efficient at around neutral pH, for this reason the Fastin Dye reagent is supplied in a buffered form. The elastin precipitating reagent (see step 7 of the protocol) has a low pH, if this is not properly removed the residual fluid can significantly alter the pH of the sample/dye mixture. This causes a change in the dye color (from red to green), and a reduction in elastin binding.

Therefore, please ensure that as much fluid is removed as possible at step 7, prior to addition of the Fastin Dye Reagent.

Why might my standard curve exhibit unexpectedly low absorbance readings?

Please first check that you have correctly prepared the assay standards. We would suggest running a standard curve using 0 - 80 micrograms of elastin standard per tube, equivalent to 80 microliters of the provided elastin standard.

Check that you are removing as much fluid possible at step 7, prior to addition of the Fastin Dye Reagent (see previous question for a more detailed explanation of why this is important).

My zero '0' value seems quite high, do you have any tips on how I can reduce the assay background?

The background absorbance can be reducing by ensuring that you remove as much excess precipitating reagent and dye as possible at steps 7 and 10 of the general protocol.