I am working on protein which binds with glycoprotein (mucin) via sugar moieties on mucin. I am interested to study protein-sugar interaction with some biophysical techniques. Can any one suggest a simple protocol for separation of sugars from mucin. Especially I want to do ITC, fluorescence and co-crystallization with the complex sugar separated from mucin with my protein of interest. Any help is highly appreciated.
Check if these links are useful
http://www.ncbi.nlm.nih.gov/pubmed/6625884
http://www.ncbi.nlm.nih.gov/pubmed/7126644
http://www.sciencedirect.com/science/article/pii/0304416582902938
http://www.sciencedirect.com/science/article/pii/S0021967301853899
http://glycob.oxfordjournals.org/content/8/11/1045.long
Following papers should give you all the info you need for the release and characterization of mucin type glycans.
K. R. Anumula. (2008) Unique Anthranilic Acid Chemistry Facilitates Profiling and Characterization of Ser/Thr Linked Sugar Chains Following Hydrazinolysis. Anal. Biochem. 373, 104–111.
K. R. Anumula. (1994) Quantitative Determination of Monosaccharides in Glycoproteins by High Performance Liquid Chromatography with Highly Sensitive Fluorescence Detection. Anal. Biochem. 220, 275-283.
K. R. Anumula. (1995) Rapid Quantitative Determination of Sialic Acids in Glycoproteins by High Performance Liquid Chromatography with a Sensitive Fluorescence Detection. Anal. Biochem. 230, 24-30.
K. R. Anumula. (2014) Single Tag for Total Carbohydrate Analysis. Anal. Biochem. 457, 31-37.
These are the industry std. methods and you will find some improvement in the HPLC separations using new columns from different vendors.
Perhaps, you can use 2-AA labeled glycans for interaction studies by fluorescence.
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