Analysis of ADisaccharides Using Ultraviolet Detection

Following digestion of samples containing CS and/or DS with both chondroitinases ABC and AC, all constituent nonsulfated and variously sulfated disaccharides of these

Fig. 3. Electropherograms showing the resolution of GalAG-derived A-disaccharides following degradation of CS/DS with chondroitinases ABC and AC. (A) Direct UV detection at 232 nm and (B) derivatization of the A-disaccharides obtained with fluorescent tag 2-aminoacridone and detection using LIF (Xexc 488 nm). In both cases, analysis is performed using 15 mM phosphate buffer, pH 3.0. Peaks: 1 = Adi-tri(2,4,6)S; 2 = Adi-di(2,6)S; 3 = Adi-di(2,4)S; 4 = Adi-di(4,6)S; 5 = Adi-mono2S; 6 = Adi-mono4S; 7 = Adi-mono6S; 8 = Adi-nonSCS/DS; 9 = Adi-nonSHA. Reprinted from refs. 1 and 2, copyright 1995, 1999 Elsevier Science, with permission.

Migration time (min)

Fig. 3. Electropherograms showing the resolution of GalAG-derived A-disaccharides following degradation of CS/DS with chondroitinases ABC and AC. (A) Direct UV detection at 232 nm and (B) derivatization of the A-disaccharides obtained with fluorescent tag 2-aminoacridone and detection using LIF (Xexc 488 nm). In both cases, analysis is performed using 15 mM phosphate buffer, pH 3.0. Peaks: 1 = Adi-tri(2,4,6)S; 2 = Adi-di(2,6)S; 3 = Adi-di(2,4)S; 4 = Adi-di(4,6)S; 5 = Adi-mono2S; 6 = Adi-mono4S; 7 = Adi-mono6S; 8 = Adi-nonSCS/DS; 9 = Adi-nonSHA. Reprinted from refs. 1 and 2, copyright 1995, 1999 Elsevier Science, with permission.

GAGs are recovered as A-disaccharides. All CS/DS-derived A-disaccharides are completely resolved and determined using direct UV detection with the following protocol.

1. Dissolve the samples containing approximately 0.1-10 ^g of CS and/or DS in 10 ^L of digestion buffer I.

2. Add 40 ^L of GalAGs degradation buffer to the samples and digest for 90 min at 37°C (see Note 7).

3. Centrifuge the digestion mixture in a microfuge tube at 11,000g for 5 min (see Note 8).

4. Place aliquots of the supernatant in the electrophoresis vials and keep at 4°C pending use.

5. Start the CE instrument, adjust the detector wavelength at 232 nm, prepare to use the method and analyze the samples, as described under Subheading 3.1, steps 6-8. (see Fig. 3A).

Was this article helpful?

0 0
Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook


Post a comment