By Adam P. Arkin
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Extra info for Current Protocols in Chemical Biology 2010 (Volume 2)
Additionally, the following protocol describes quality-control checks for evaluating array slides. com). ch090228 Copyright C 2010 John Wiley & Sons, Inc. 37 Volume 2 Neoglycoproteins Acquisition of a diverse set of glycans and carbohydrates is a key step for construction of a glycan array. The focus of this protocol is on the production of neoglycoprotein arrays. Neoglycoproteins are non-naturally-occurring conjugates formed by covalently linking a glycan to a carrier protein, such as albumin.
5 g (21 mmol)] and stir under a reflux condenser for 12 hr. 3. Remove solvent under reduced pressure in Buchi Rotovapor. 4. Add 150 ml of ethanol and place at 4◦ C for 5 hr. 5. Filter the crystals and wash with cold ethanol to obtain N6 phenethyl (or additional alkyl) adenosine. Synthesize N6 phenethyl ADP 6. 76 g) in 5 ml of triethethylphosphate (TEP). 7. Cool to 0◦ C in an ice/NaCl bath while stirring. 8. 6 g (4 mmol) of POCl3 dropwise and continue to stir at 0◦ C for 2 hr. 9. 0]undec-7-en (DBU) in 5 ml TEP, then add 8 mmol of H3 PO4.
Thiophosphorylated proteins are alkylated, and the candidate substrate is immunoprecipitated using an antibody against that protein. If a band is detected by immunoblotting with thiophosphate ester rabbit monoclonal antibody, then the candidate is a substrate of the KOI. 5-ml microcentrifuge tubes Additional reagents and equipment for SDS-PAGE and immunoblotting (Support Protocol 1) 1. Grow appropriate mammalian cells to 60% to 80% confluence on 10-cm dishes. 2. Transfect with plasmid encoding wild-type or AS KOI using transfection reagent of choice, following the manufacturer’s protocol.