From sample preparation to setting up a standard curve, here we answer some of the most common questions relating to our assay kits.
Please contact us if you have further questions, we enjoy hearing from our customers and our technical team will get back to you shortly!
Q. How long does the Sircol 2.0 assay take to run?
A. The total assay time (assuming samples are ready to analyse) is approximately 4-hours, allowing analysis to be completed inside one working day.
Q. What forms of collagen does the Sircol assay measure?
A. Acid-soluble and pepsin-soluble collagens.
Q. What types of collagen can the Sircol assay be used with?
A. Mammalian collagens, Types I to V.
Q. How do I prepare samples for measurement with the Sircol 2.0 assay?
A. Preparation details for cell culture and tissue samples can be found in Part D of the assay manual.
Q. What sort of collagen standard should I use?
A. The Kit containing bovine collagen is suitable for use with all mammalian-derived samples. Samples from other species should ideally use a species-matched, purified preparation of collagen as a standard (see p6 of manual).
Q. What absorbance values can samples be measured at?
A. Absorbance spectrum maximum for Sircol 2.0 dye in alkali reagent is 556 nm, however absorbance settings within the range 530-560 nm will give suitable readings.
Q. What is the sensitivity of the APOPercentage assay?
A. A single apoptotic cell within a microwell cell population (when using Digital Image Analysis)
Q. How long does the APOPercentage assay take to run?
A. 1 hour.
Q. What types of cell can the assay be used with?
A. Established cell lines are suitable. Cells should be anchorage-dependent. In order to enhance attachment cells exposed to toxic agents may be grown in gelatin.
Q. What absorbance values can samples be measured at?
A. 550nm on a microplate colorimeter.
Q. What is the difference between the S1000 and Sircol 2.0 assay kits?
A. The S1000 was Biocolor’s original soluble collagen assay. It utilized samples processed within 1.5ml microtubes, followed by manual transfer to a microplate for spectrophotometric analysis. The Sircol 2.0 kit is its modern successor, based on an improved dye formulation, combined with the processing and measurement of samples directly within the convenient microwell plate format. As a result, Sircol 2.0 is more user-friendly, while also offering increased collagen sensitivity and specificity.
Q. Is the S1000 kit still on the market?
A. Biocolor will continue to offer the S1000 kit and related consumables for sale. We are pleased to continue to support our wide range of commercial and academic customers who have incorporated the S1000 kit into their laboratory workflows.
Q. What is the sensitivity of the Sircol assay?
A. 1.0µg collagen per assay tube, equivalent to 10µg/ml. Quantities as low as 1µg of collagen can be recovered from a 1 ml volume if the Collagen Isolation & Concentration protocol is used (See Sircol assay manual for more detail).
Q. How long does the Sircol assay take to run?
A. 1.5 hours
Q. What forms of collagen does the Sircol assay measure?
A. Acid-soluble and pepsin-soluble collagens.
Q. What types of collagen can the Sircol assay be used with?
A. Mammalian collagens, Types I to V.
Q. What sort of collagen standard should I use?
A. The Kit containing bovine collagen is suitable for use with all mammalian cells.
Q. What absorbance values can samples be measured at?
A. Absorbance spectrum maximum for Sircol dye in alkali reagent is 556 nm, however absorbance settings within the range 520-570 nm will give suitable readings.
Q. How do I prepare samples for measurement with the Sircol assay?
A. Preparation details for cell culture and tissue samples can be found in the assay manual.
Q. What absorbance values can samples be measured at?
A. Absorbance spectrum maximum for Sircol dye in alkali reagent is 556 nm, however absorbance settings within the range 520-570 nm will give suitable readings.
Q. Why does the Sircol Insoluble collagen assay demonstrate lower aborbance values when compared to the Sircol Soluble collagen assay?
A. Maximal binding of Sircol dye occurs in collagens possessing intact triple helix organisation since the highly ordered [Gly-X-Y]n helical structure of tropocollagen contributes to dye binding. Affinity is progressively reduced during heat denaturation (>45°C) due to the unwinding of the triple helix and formation of random chains. Since samples and standards for the Sircol Insoluble collagen assay are in a denatured state (necessary to convert insoluble 'solid' samples into the liquid phase for assay) they will therefore bind less dye than the same amount of material in the non-denatured state.
Q. The collagen pellet is much smaller than the pellets in the S1000 Soluble collagen assay, why is this?
A. Samples in the Sircol Insoluble collagen assay are gently denatured via treatment with Sircol Fragmentation Reagent. This denaturation causes some loss of collagen structure. The dye-bound pellet will therefore be more 'dense' as it is able to pack more tightly following centrifugation.
Q. What is the sensitivity of the Hyaluronan assay?
A. 0.2μg in 20μl of assayed sample, equivalent to 10μg/ml.
Q. What absorbance values can samples be measured at?
A. The absorbance peak for Hyaluronic Acid in Purple-Jelley dye reagent is 655nm.
Q. How do I prepare samples for measurement with the Hyaluronic Acid assay?
A. 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).
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 forTPPS in the dye dissociation reagent is 513nm. However, absorbance settings of545nm or 550nm also give usable 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. SAMPLE PROCESSING: 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 & conversion to soluble α-elastin or soluble κ-elastin is given (see page 4of assay manual). After solubilisation the samples are assayed by the procedure described for soluble elastin.
Q. SAMPLE PROCESSING: My sample appears to be undigested following oxalic acid extraction, what can I do?
A. The product manual suggests 60 minutes 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 3hours or even overnight. Please note that multiple extractions may be required, see p4 of manual for details.
A. 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 lyophilisation or snap freezing and grinding of the material in the frozen state. Such material should extract more quickly.
Q. ASSAY METHODOLOGY: Why are my standards or samples stained green (rather than red/brown)?
A. 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 colour (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.
Q. ASSAY METHODOLOGY: Why might my standard curve exhibit unexpectedly low absorbance readings?
A. Please first check that you have correctly prepared the assay standards. We would suggest running a standard curve using 0 – 80 micrograms (ug) of elastin standard per tube, equivalent to 80 microlitres (ul) of the provided elastin standard.
A. Check that you are removing as much fluid as possible at step 7, prior to the addition of the Fastin Dye reagent (see previous question for a fuller explanation why this is important).
Q. ASSAY METHODOLOGY: My zero '0' value seems quite high, have you any tips on how I can reduce the assay background?
A. The background absorbance can be reduced by ensuring that you remove as much excess precipitating reagent and dye as possible at steps 7 and 10 of the general protocol.
Q. How long does the Fastin assay take to run?
A. 4 hours (following sample extraction).
Q. How stable is the Fastin assay?
A. Unopened, all the reagents are stable for 1 year at room temperature. After opening, follow storage conditions indicated on each component label. Do not freeze kit or remove metal seal from reference standard until ready to use.
Q. What is the sensitivity of the Blyscan assay?
A. 0.25μg/100μl, equivalent to 2.5μg/ml
Q. How long does the Blyscan assay take to run?
A. 1 hour
Q. Can I differentiate between N-sulfated and O-sulfated glycosaminoglycans?
A. A method is given in the manual (see pages 9-10) to differentiate between the glycosaminoglycans present. Supporting reagents are supplied in the kit (with exception of Heparin Sulfate, used as a comparitive standard).
Q. What absorbance values can samples be measured at?
A. The absorbance peak for Blyscan dye in the dissociation reagent is 656nm. This absorbance is suitable for use with most colorimeters and microplate readers with a red filter.
Q. How do I prepare samples for measurement with the Blyscan assay?
A. Sulfated GAGs (sGAG) should be in soluble form for use in the Blyscan assay. Preparation details for samples in tissue culture medium, cartilage/soft tissue and urine samples are described in the assay manual. Many samples will require solubilisation of the sGAG using an eznzymatic pre-treatment, see our FAQ regarding this step:
Q. Why do I have to use Papain enzyme for sample extraction, and where can I get it from?
A. Sulfated GAGs (sGAG) should be in soluble form for use in the Blyscan assay. The best way to achieve this is to perform sGAG extraction using the enzyme – Papain. Since Papain is unstable in its active form, we cannot ship it with the kit. It must therefore be purchased in its ‘inactive’ form, which is then activated by adding it to a buffer (preparation instructions are on p3 of the manual, or in the question below.
Recommended Enzyme:
We recommend customer purchase ‘Papain from papaya latex’ (product code P3125), from Merck:
https://www.sigmaaldrich.com/GB/en/product/sigma/p3125
A suitable alternative would also be the Papain suspension (product code PAP) by Worthington Biochemical:
http://www.worthington-biochem.com/pap/cat.html
Q. I have the papain enzyme, how do I prepare the Papain sample extraction buffer?
A.Please follow the steps below:
Preparation of enzyme and buffer for use with samples
The papain enzyme (such as the Merck p3125) will be supplied as an aqueous suspension. It should be supplied with a label or datasheet to say how much actual enzyme is dissolved per unit volume -this may vary from batch to batch. We suggest using about 5mg of the active papain. Due to batch-to-batch variation the volume required to do this can therefore vary. You will need to check this datasheet and then calculate what volume of papain suspension will be required to contain 5mg of the enzyme.
The papain suspension will not digest any protein until it is first activated – this is achieved by diluting into a phosphate buffer containing the necessary cofactors. The buffer recipe is provided on p3 of the manual.
Step1: Prepare the phosphate buffer at the correct pH, the following website may be helpful:
https://www.aatbio.com/resources/buffer-preparations-and-recipes/phosphate-buffer-ph-5-8-to-7-4
A 50 ml volume of this will then be set aside, the cofactors and enzyme will be added to this 50ml volume.
Step 2: Add 400 mg sodium acetate
Step 3: Add 200 mg EDTA, disodium salt
Step 4: Add 40 mg cysteine HCl
Step 5: Stir to dissolve all components.
Step 6: Then add your calculated amount of the Papain suspension (see above), make sure you have calculated the volume so that you are adding a total of 5 mg of active enzyme.
Step 7: Stir to dissolve.
This can then be used with your sample.
Q. Can the assay be used on urine, synovial fluid or other water-based samples?
A. Yes, the assay can be used with such samples, further guidance is found on p6 of the manual.
Please note, since these samples are prone to variable concentration and composition we have developed an additional 'Blyscan sGAG Isolation & Concentration Pack' - product code: B1015 (100 samples) which can aid sample preparation.
Please contact us for further details.
Q. How can I prevent dye-labelled glycosaminoglycan pellets falling or sliding out of the tube when I am removing the unbound dye?
A. This can be minimised by modifying the previous centrifugation step as follows: First perform the usual spin at 13000 x g for 10 minutes. Then allow the centrifuge to stop. Wait for 2 – 3 minutes then proceed with a second 13000 x g centrifugation for a further 5 minutes. This should improve the adhesion of the dye-labelled glycosaminoglycan containing pellets to the tubes.