Immunoblotting Assays for Keratan Sulfate Jung Hae Yoon, 1 Randolph Brooks, and Jaroslava Halper The Soft Tissue Center, Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602 Received December 27, 2001; published online June 18, 2002 The detection of microquantities of glycosaminogly- cans (GAGs) in biological samples has been hampered by the lack of sensitive methods. In this paper we describe the modification and development of three sensitive as- says capable of detecting nanogram quantities of GAGs in biological samples. The first assay detects total GAGs. It is a modified Alcian blue dye precipitation assay in which the dye binds to the negatively charged GAGs in CsCl-fractionated extracts from chicken tendons. This assay compares favorably with the widely used uronic acid assay in terms of its sensitivity and ability to detect all classes of GAGs, including keratan sulfate (KS). Two other assays, dot-blotting and immunoblotting, detect KS in complex mixtures and can be easily adapted for the detection of other GAGs. Both take advantage of binding of carboxyl and sulfate groups of GAGs to trivalent neodymium. In dot-blotting, samples were di- rectly blotted onto nitrocellulose membrane soaked in Nd 2 (SO 4 ) 3 buffer, and KS was detected with the mono- clonal anti-KS 5-D-4 antibody and an avidin– biotin com- plex detection system. In immunoblotting, the samples were first separated in 28% polyacrylamide gels, trans- ferred onto a Nd 2 (SO 4 ) 3 -soaked nitrocellulose membrane using a phosphate buffer system, and stained and devel- oped using the same protocol as in dot-blotting. Whereas dot-blotting allows the use of very low quantities of sam- ples because of its high sensitivity (lower detection limit was 5 ng), immunoblotting provides more specificity. © 2002 Elsevier Science (USA) Key Words: dot-blotting; glycosaminoglycans; immu- noblotting; keratan sulfate; Nd 2 (SO 4 ) 3 . Proteoglycans (PGs) 2 consist of core proteins to which glycosaminoglycans (GAGs), polyanionic poly- saccharides composed of repeating disaccharide units, are covalently linked (1). GAGs can be divided into four classes based on the composition of their disaccharide units, including the presence of sulfate or carboxyl groups: (1) heparin and heparan sulfate (HS), (2) chon- droitin sulfate (CS) and dermatan sulfate, (3) hyal- uronic acid (HA), and (4) keratan sulfate (KS). KS is the only class of GAGs that does not have uronic acid in its structure (2). In most cases of KS, the hydroxyl group at the C-6 position of the GlcNAc residue is sulfated. The degree of sulfation varies between 0.4 and 1.5 per disaccharide unit, depending on the tissue or organ in which KS is found; different sulfation de- grees have been observed in the cornea (3), various types of cartilage (4 – 6), and the brain (7, 8). The degree of the sulfation of KS changes with dis- ease and during normal physiological development. It has been shown to increase in corneal cataracts (9) and in degenerative disc disease (10). The degree of sulfa- tion of KS has also been shown to change in embryonic development (11, 12) and the regeneration of the endo- metrium during the menstrual cycle (13). Progress in these studies has been hampered by the relative lack of sensitive methods for GAG detection and identifica- tion. Several types of detection methods for GAGs are in use. The most widely used is a uronic acid assay (14). However, because it is based on the colorimetric detec- tion of uronic (both glucuronic and iduronic) acid units, this assay does not identify GAGs, like KS, that lack uronic acid. Other detection methods for GAGs take advantage of the polyanionic nature of the carbohy- drate chains in biological samples. In particular, the precipitation of GAGs with bivalent cations, such as Ba 2+ (15), Ca 2+ (16), and Cu 2+ (17), or with cationic dyes, such as safranin O (18), Alcian blue (19), and dimethylmethylene blue (20), has been important in GAG analysis. The disadvantage of these agents is that they may bind with other negatively charged com- pounds. Recently, liquid chromatography/turbo-ion- spray tandem mass spectrometry has also been em- ployed (21). Though this technique is very powerful, 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (706) 542-5828. E-mail: jyoon@vet.uga.edu. 2 Abbreviations used: GAG, glycosaminoglycan; KS, keratan sul- fate; ELISA, enzyme-linked immunosorbent assay; PVDF, poly- vinylidene difluoride; CS, chondroitin sulfate; HS, heparan sulfate; HA, hyaluronic acid; Me 2 SO, dimethyl sulfoxide; Chase, chondroiti- nase; PG, proteoglycan; TPCK, L-1-tosylamide-2 phenylethyl chlo- romethyl ketone; CAPS, 3-cyclohexylaminol-1-propane sulfomic acid. 298 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. Analytical Biochemistry 306, 298 –304 (2002) doi:10.1006/abio.2002.5711