Blyscan™ sGAG (Glycosaminoglycan) assay kit

0.25μg/100μl, equivalent to 2.5μg/ml

1 hour

Yes. The kit includes reagents to separate N-sulfated GAGs (heparin and heparan sulfate) from O-sulfated GAGs (such as chondroitin, dermatan and keratan sulfates).

Sodium nitrite and acetic acid are supplied with the kit. These form nitrous acid, which cleaves the GAG chain at N-sulfated glucosamine residues, removing the dye-reactive sulfate groups. After treatment, Blyscan therefore measures only the O-sulfated GAG.

The method requires a heparin or heparan sulfate standard (not included with the kit). This is used in combination with the assay standard to prepare a comparative calibration curve (comprising known mixtures of purified N- and O-sulfated GAGs).

In outline:

1. Mix 100 µl of sample with 100 µl of sodium nitrite and 100 µl of acetic acid.
2. Leave for 60 minutes at room temperature.
3. Add 100 µl of ammonium sulfamate to remove excess nitrous acid. Expect some bubbling.
4. Mix for 10 minutes, then assay 100 µl of the mixture as normal.

The results are calculated as follows:

• O-sulfated GAG is the result for the treated sample, allowing for the four-fold dilution.
• N-sulfated GAG is total sGAG (the untreated sample) minus O-sulfated GAG.

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.

Blyscan measures sulfated glycosaminoglycans (sGAG) in solution. Clear liquids can usually be assayed directly. Tissues and cell layers first need an enzyme digest to release their sGAG in a soluble form.

• Clear liquids (culture medium, urine, synovial fluid): remove any cloudiness or particles by centrifugation, then assay.
• Synovial fluid: viscous samples can be diluted in PBS for accurate pipetting. The hyaluronan in synovial fluid doesn't interfere.
• Urine: an early-morning sample is best. Assay two volumes (25 µl and 100 µl) and express results per mmol of creatinine or per 100 ml.
• Tissues, cartilage and bone: digest 20–50 mg of tissue in 1 ml of papain extraction reagent at 65 °C. Soft tissue takes about 3 hours; cartilage and bone need overnight. Centrifuge at 10,000 x g for 10 minutes and assay the supernatant.
• Cell cultures: collect the medium, then digest the cell layer in papain reagent at 65 °C for 3 hours. Total sGAG is the medium and the digest added together. The manual also describes how to measure the cell-surface, matrix and cell-content fractions separately.

Papain isn't supplied in the kit as the active enzyme isn't stable in transit. See the papain questions below for where to purchase it and how to prepare the papain extraction buffer.

If samples contain a lot of salt or detergent, prepare the standard curve in the same diluent. For low or variable samples, the Blyscan sGAG Isolation & Concentration Pack (B1015) can be used. Contact us for further information.

Full protocols are in the Blyscan manual. You can download it from the Documentation links at the top of this page.

Sulfated GAGs (sGAG) must be in soluble form for Blyscan analysis. A convenient means to release them from solid samples is digestion with papain. Active papain isn't stable in transit, so we can't ship it with the kit. It is generally purchased in an inactive form and activated by diluting it into a phosphate buffer containing cysteine and EDTA (see the next question for how to prepare it).

Recommended enzyme:

We recommend customers 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

Papain is supplied as an inactive suspension. It only becomes active once it's diluted into a phosphate buffer containing cysteine, which activates the enzyme, and EDTA. Make up the papain extraction reagent as follows:

1. Prepare 50 ml of 0.2 M sodium phosphate buffer, pH 6.4. This phosphate buffer guide may help.
2. Add 400 mg sodium acetate.
3. Add 200 mg EDTA, disodium salt.
4. Add 40 mg cysteine HCl.
5. Stir until everything has dissolved.
6. Add papain suspension to give about 5 mg of active enzyme. With Merck P3125 this is typically around 250 µl. The enzyme concentration varies from batch to batch, so check the datasheet supplied with your enzyme and calculate the volume you need.
7. Stir to mix.

The reagent keeps for up to 7 days at 4 °C. Use it at 65 °C as described in the sample preparation question above.

Yes. Clear, water-based samples such as urine and synovial fluid can usually be assayed directly, without an enzyme digest.

• Cloudy samples: remove any cloudiness or particles first by centrifugation.
• Synovial fluid: dilute viscous samples in PBS so they can be pipetted accurately. Hyaluronan doesn't interfere with the assay.
• Urine: an early-morning sample is best. Assay two volumes (25 µl made up to 100 µl with water, and 100 µl undiluted). Urinary sGAG levels vary widely, so this makes it more likely that one reading falls within the standard curve. If both give the same result once you allow for the dilution, you can also be confident the urine isn't interfering with the assay. Express results per mmol of creatinine or per 100 ml.
• High salt or detergent: if samples contain either, prepare the standard curve in the same diluent.

Since these samples often vary in concentration and composition, we have developed the Blyscan sGAG Isolation & Concentration Pack (B1015, 100 samples), which can help with sample preparation. Contact us for details.

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.