Biocolor Blyscan™ Glycosaminoglycan (sGAG) Assay - Frequently Asked Questions (FAQ)

Here you will find answers to many of the frequently asked questions about Biocolor Blyscan™ Glycosaminoglycan (sGAG) Assay.

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What is the Blyscan™ Glycosaminoglycan (sGAG) Assay?

The Biocolor Blyscan™ Glycosaminoglycan (sGAG) Assay utilizes a colorimetric dye-binding approach to quantitatively measure sulfated glycosaminoglycans (sGAG) and proteoglycans in cells, tissues, and fluids from in-vivo and in-vitro sources.

General Questions

What is the sensitivity of the Blyscan™ sGAG Assay?

0.25 µg/100 µl, equivalent to 2.5 µg/ml.

How long does Blyscan™ sGAG Assay take to run?

1 hour

Can I differentiate between N-sulfated and O-sulfated glycosaminoglycans?

Yes, 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 the exception of Heparin Sulfate, used as a comparative standard).

What absorbance values can samples be measured at?

The absorbance peak for Blyscan Dye in the dissociation reagent is 656 nm. This absorbance is suitable for use with most colorimeters and microplate readers with a red filter.

Sample Processing

How do I prepare samples for measurement using the Blyscan™ sGAG Assay?

Sulfated GAGs (sGAG) should be in the 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 solubilization of the sGAG using an enzymatic pretreatment (see more details in the next question).

Why do I have to use Papain enzyme for sample extraction, and where can I get it from?

Sulfated GAGs (sGAG) should be in the soluble form for use in the Blyscan Assay. The best way to achieve this is to perform sGAG extraction using papain enzyme. Since papain is unstable in its active form, we cannot ship it with the kit. Therefore it must be purchased in its "inactive" form, which is then activated by adding it to a buffer (preparation instructions are on page 3 of the manual, or in the question below).

Recommended Enzyme:

We recommend that the customer purchase "Papain from papaya latex" (product code P3125), from MilliporeSigma.

A suitable alternative would be the Papain Suspension (product code: PAP), from Worthington Biochemical.

I have the papain enzyme, how do I prepare the papain sample extraction buffer?

Please follow the steps below:

Preparation of enzyme and buffer for use with samples:

The papain enzyme 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 5 mg of the active papain. Due to batch-to-batch variation, the volume required to do this may vary. You will need to check the datasheet and then calculate what volume of papain suspension will be required to contain 5 mg 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 page 3 of the manual.

Step 1: Prepare the phosphate buffer at the correct pH. A 50 ml volume of this will then be set aside, the cofactors and enzyme will be added to this 50 ml volume.

Step 2: Add 400 mg sodium acetate.

Step 3: Add 200 mg EDTA, disodium salt.

Step 4: Add 40 mg cysteine HCI.

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.

Can the assay be used on urine, synovial fluid, or other water-based samples?

Yes, the assay can be used with such samples, further guidance is found on page 6 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.

Assay Methodology

How can I prevent dye-labeled glycosaminoglycan pellets falling or sliding out of the tube when I am removing the unbound dye?

This can be minimized by modifying the previous centrifugation step as follows: First perform the usual spin at 13000 x g for 10 minutes. The allow the centrifuge to stop. Wait 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-labeled glycosaminoglycan containing pellets to the tubes.