[ Pre-treatment Procedure ]

Different preprocessing methods are used for different types of test sample. Be careful to use the correct method.

Tissue Samples (a)

1. Collect 1- 100mg of tissue samples.
2. Add 1,800µL of Protease Diluent and 200µL of Protease Solution and then mix for 10sec.
3. Incubate for 20hrs. at 55°C and then boil for 10min. and cool to room temperature.

Cultured Chondrocytes (b)

1. Collect 1- 8×10⁵ cells of cultured chondrocyte.
2. Add 180µL of Protease Diluent and 20µL of Protease Solution and then mix for 10sec.
3. Incubate for 2hrs. at 55°C and then boil for 10min. and cool to room temperature.

Cell Culture Supernatant (c)

1. Collect 180µL of Culture Supernatant.
2. Add 20µL of Protease Solution and then mix for 10sec.
3. Incubate for 2hrs. at 55°C and then boil for 10min. and cool to room temperature.

[ Assay Procedure ]

Verify all reagents and samples are at room temperature (15-25°C) before commencing assay.
It is recommended that all standards and samples be assayed in duplicate.

1. Prepare all reagents, Sulfated GAG Standard Solutions and samples.^*1
2. Prepare Microplates (A).
3. Add 50 µL of Sulfated GAG Standard Solution (80, 40, 20, 10, 5, 2.5 ?g/mL), blanks (0 µg/mL), and/or protease-treated samples.
4. Note: Different methods are used for different types of test sample. Be careful to use the correct method.
   
   • Tissue Samples(a): Add 50 µL of Reaction Buffer I (D) to each well.^*2
   • Cultured Chondrocytes (b) and/or Cell Culture Supernatant (c): Add 50 µL of Reaction Buffer II (E) into each well.^*2
5. Add 150 µL of DMMB Dye Solution (C) to each well.^*2
6. Incubate for 5 min. at room temperature (15-25°C) in the dark by covering the whole strips using aluminum foil.
7. Measure absorbance at 530nm using a microplate reader immediately.
   *1 Remove particulates by centrifugation, and dilute with Sample Diluent if needed.
   *2 Important: Multidispense pipette is recommended for accurate assay.

[ Calculation of Sulfated GAG concentration ]

1. Average duplicate values for each Sulfated GAG standard and sample.
2. To make the Sulfated GAG standard curve, plot the Sulfated GAG concentration on the x-axis and the absorbance on the y-axis for each standard Sulfated GAG solution using graph paper or appropriate software. We recommend to use [quadratic] curve for the plot.
3. Determine the Sulfated GAG concentration for each diluted sample using the generated Sulfated GAG standard curve.
4. To obtain the Sulfated GAG concentration in each sample, multiply the above value by the appropriate dilution factor.
   *As to (diluted) samples that exceeded 80µg/mL, repeat the assay with proper dilution in Sample Diluent.

[ Typical standard curve ]

1. Plot an x-y quadratic curve, with concentration in mg/mL on the x-axis and absorbance on the y-axis.
2. Calculate the inverse function and substitute the absorbance on the y-axis.
3. Convert the x value thus obtained to µg/mL.
   *An Excel conversion sheet is available. Please click here to contact us if you would like to request a copy..