Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications

Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. al. 2006; Whitworth et al. 2007; Yuzwa et al. 2008), although only PUGNAc (Toronto Study Chemicals; (Haltiwanger et al. 1998) and Thiamet-G (Cayman Chemicals; (Yuzwa et al. 2008)) are commercially available. Unlike Thiamet-G, PUGNAc also inhibits lysosomal hexosaminidases. Streptozotocin (STZ; Roos et al., 1998), glucosamine (Han et al., 2000), and the glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitors 6-diazo-5-oxonorleucine (DON) and Azaserine have also been used to alter the stoichiometry of O-GlcNAc on proteins. However, STZ offers been shown to induce poly-(ADP-ribose) polymeraseCmediated apoptosis in Min6 cells (Gao et al., 2000) and should be used with caution. In addition, STZ is only effective in cells that communicate the glucose transporter GLUT-2 (Schnedl et al., 1994). Recently, several specific OGT inhibitors have been developed (Gross et al. 2005). These inhibitors work well DETECTION OF PROTEINS MODIFIED BY O-GlcNAc USING ANTIBODIES Several antibodies have been developed that identify terminal GlcNAc residues (DETECTION OF PROTEINS Procyanidin B3 MODIFIED BY O-GLcNAc USING THE Procyanidin B3 LECTIN sWGA Many lectins are reportedly specific for -GlcNAc residues. The authors have typically used succinylated wheat germ agglutinin (sWGA), which is definitely widely available and is derivatized with a number of useful functional organizations including horseradish peroxidase (HRPO). Before succinylation, WGA will recognize both silaic acid and GlcNAc (Monsigny et al. 1980). For additional information concerning lectin chromatography, observe CONTROL FOR O-LINKED GLYCOSYLATION Traditionally, mild alkaline reduction (reductive -removal) has been used to release O-linked carbohydrates from proteins (Amano and Kobata 1989). This method has been adapted for blots to show that lectin/antibody reactivity is definitely toward O-linked rather than N-linked glycans (Duk et al. 1997). Proteins blotted to PVDF are treated with 55 mM NaOH over night (liberating O-linked sugars) and then probed using lectins or antibodies. There are a number of reasons why lectin/antibody reactivity could be lost after NaOH treatment, e.g., the sugars were damaged instead of becoming released, or the protein was degraded. To control for these, it is important to have control proteins with N- and O-linked sugars, and to stain one blot for protein after treatment preferably with an antibody. The authors suggest a control blot of bovine asialofetuin (Sigma) which consists of both N- and O-linked sugars terminating in GlcNAc, treated and not treated with PNGase F (DETECTION AND ENRICHMENT Procyanidin B3 OF PROTEINS USING WGA-AGAROSE WGA lectin affinity chromatography provides a convenient method for enriching and detecting O-GlcNAc revised proteins. This procedure has been adapted for detecting proteins that are hard to purify or are present in low copy number, such as transcription factors. With this protocol, the protein of interest is definitely synthesized inside a rabbit reticulocyte lysate (RRL) in vitro transcription translation (ITT) system (Promega) and MSK1 labeled with either [35S]Met, [35S]Cys, or [14C]Leu. After desalting, the proteins are tested for his or her ability to bind WGA agarose inside a GlcNAc-specific manner (Roquemore et al. 1994). This protocol is definitely readily adapted to purifying proteins from cell components, but, as WGA binds proteins with both terminal GlcNAc and SILAC acid residues typically one would purify proteins from nuclear and cytoplasmic components to avoid co-purifying proteins with proto-typical glycans. On Procyanidin B3 the other hand, the lectin agglutinin 1 (RCA1) has been used to select for O-GlcNAc proteins that have previously been labeled by galactosyltransferase (observe Alternate Protocol 1). Proteins revised by terminal Gal are specifically retained on a RCA1 affinity column. Labeled O-GlcNAc proteins are released under slight conditions, while those comprising N-linked structures require lactose addition to the buffer before elution results (Greis and Hart 1998; Hayes et al. 1995). The method described with this protocol can be.

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