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. 2017 Nov 1;7:14785. doi: 10.1038/s41598-017-15009-0

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Sequencing of chondroitin sulfate from the proteoglycan bikunin and (bio)availability of sequence-defined fragments. (a) BCN end-labeled chondroitin sulfate digested with chondroitinase AC-II, separated by PAGE and blotted to tetrazine-functionalized paper followed by incubation with the antibody 1B5. From the staining pattern the sequence can be read (arrow: OS disaccharide close to reducing end; square bracket: the transition zone). (b) Same as (a) but azidoaniline end-labeling and DBCO-functionalized blot paper. 1B5 and 2B6 stainings are on different lanes. Long and short run: electrophoresis for 5.5 and 3.5 h, respectively. (c) Identification of the first disaccharide. BCN-labeled chondroitin sulfate digested with chondroitinase ABC, subjected to PAGE, and blotted to tetrazine-functionalized blot paper followed by staining with antibody 2B6. A non-stained fragment was cut out of the blot paper and digested with chondroitinase AC-II releasing the first disaccharide which was identified as ∆UA-GalNAc4S by RP-HPLC (right picture, see arrow). (d) Identification of uronic acid moiety. Azidoaniline end-labeled chondroitin sulfate digested with hyaluronidase with or without exo-glucuronidase. The shift in migration identifies the uronic acid as glucuronic acid. (e,f) In situ (bio)availability of sequence-defined chondroitin sulfate fragments. (e) BCN end-labeled chondroitin sulfate was fragmented as under (a) and visualized using antibody 2B6. Non-staining fragments became apparent after additional on-blot digestion with chondroitinase ABC + AC, creating new antibody- 2B6 positive bands (see e.g. lanes labeled 5′). (f) Chemical availability of chondroitin sulfate fragments. Azidoaniline (AA) labeled chondroitin sulfate A and decasaccharides were immobilized on DBCO-functionalized blot paper and treated without (−, upper part) or with ( + , middle part) Hg-acetate to remove the terminal unsaturated uronic acid residue resulting in a loss of antibody 2B6 staining. Staining is restored after additional treatment with chondroitinase ABC (lower part).