ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 344 (2005) 158–160 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.05.030 Notes & Tips The quantiWcation of chondroitin sulfates by rocket electrophoresis Jung Hae Yoon ¤ , Jaroslava Halper Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA Received 6 April 2005 Available online 13 June 2005 As described previously, we have developed a very sensitive assay of detecting nanogram quantities of kera- tan sulfate (KS) 1 by binding carbonyl and sulfate groups of glycosaminoglycans (GAGs) to trivalent neodymium [1]. KS is the basic repeating unit of GlcNAc(1,3)– Gal(1,4) and, unlike other GAGs, does not have uronic acid in the structure. Samples were transferred to a nitro- cellulose membrane soaked in Nd 2 (SO 4 ) 3 buVer, and KS was detected using the monoclonal anti-KS 5-D-4 anti- body and an avidin–biotin complex detection system. We have investigated the utility and validity of our approach by comparing KS content in young chicken gastrocnemius tendons of diVerent ages (1 day, 1 week, and 6.5 weeks) and showed that the lower detection limit was 5 ng. We have indicated that our assay could be applied to a quantitative measurement of other GAGs. In this study, we used the same principle for the quan- tiWcation of chondroitin sulfate A (CS A, Di-4S) and chondroitin sulfate C (CS C, Di-6S) with CS 56 anti- body by rocket immunoelectrophoresis. We compared the results with those obtained by HPLC previously [2]. In that study, we analyzed proteoglycan and glycosami- noglycan composition in 6.5-week-old chicken gastroc- nemius tendons. Half of the chickens underwent treadmill exercise for 4 weeks, and the control group was allowed normal movement in the holding pen [2]. The general trend of HPLC analysis of Sepharose CL-2B fractions combined from several column runs from extracted 6.5-week-old exercised and control tendons corresponded well with results obtained using rocket immunoelectrophoresis. Young female chickens were randomly divided into a control group and an exercise group. The exercise group was run on the treadmill at 1 mph for three 10-min intervals per day from 2.5 to 6.5 weeks of age. The con- trol group was allowed normal movement in holding pens, as was the exercise group when those chickens were not running on the treadmill. Gastrocnemius ten- dons were removed from 6.5-week-old chickens, divided into upper and lower halves, homogenized, and extracted with 4 M guanidine HCl, and the extracts were fractionated by ultracentrifugation in a CsCl gra- dient. Aliquots of the four bottom (dense) CsCl frac- tions from the control and exercised 6.5-week-old tendons were separated on a Sepharose CL-2B column (0.5 £ 110 cm, 0.06 ml/min, equilibrated in 4 M guani- dine HCl). Two large discernible peaks obtained from Sepharose CL-2B separation were combined and divided into Wve pools from exercised (E1–E5) material and Wve pools from control (C1-C5) material. Aliquots were treated with trypsin (1 U, bovine, Sigma, St. Louis, MO, USA), proteinase K (1 U, Tritirachium album, Sigma), and Chase AC (Proteus vulgaris) before analy- sis by HPLC (System Gold, Beckman Coulter, Fuller- ton, CA, USA) using a Partisil 5-PAC column (4 £ 250 mm, Whatman, Clifton, NJ, USA) eluted with acetonitrile/methanol (0.5 M Tris in 0.1 M boric acid (52:12:36), pH 8.0, 0.8 ml/min, at 232 nm) [1–4]. HPLC samples were analyzed in triplicate. CS A and CS C were identiWed using a monoclonal mouse anti- chondroitin antibody CS 56 (Sigma–Aldrich, St. Louis, MO, USA) diluted 1:500 and were identiWed by Coomassie blue staining. Gelbond Wlm (agarose gel sup- port medium, hydrophilic side up) was obtained from Biowhittaker Molecular Applications (Rockland, ME, USA). Rocket immunoelectrophoresis was carried out in 1% agarose (w/v) that was dissolved in 0.1 M Nd 2 (SO 4 ) 3 containing 0.1% of CS 56 antibody. After gel * Corresponding author. Fax: +1 706 5425828. E-mail address: junghae.yoon@gmail.com (J.H. Yoon). 1 Abbreviations used: KS, keratan sulfate; GAG, glycosaminoglycan; CS A, chondroitin sulfate A; CS C, chondroitin sulfate C.