NuPAGEr凝胶常见问题-NuPAGE.pdfVIP

FAQs ® NuPAGE ® NuPAGE Frequently Asked Questions (FAQs) Frequently Asked Questions What is denaturing DS? Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by “wrapping around” the polypeptide backbone. When the protein is heated to 100°C in the presence of excess SDS and thiol reagent, disulfide bonds are cleaved and the protein is fully dissociated into its subunits. Under these conditions, most polypeptides bind SDS at a constant weight ratio (1.4 g of SDS to 1 g of polypeptide). Because the intrinsic charges of the polypeptide are insignificant compared to the negative charges provided by the bound detergent, the SDS–polypeptide complexes have essentially the same negative charge and shape as unbound polypeptides and migrate through the gel strictly according to polypeptide size. What is the chemistry of the The NuPAGE® Bis-Tris discontinuous buffer system involves three ions: ® — Chloride (Cl–) from the gel buffer serves as a leading ion. The gel buffer ions are Bis-Tris (+) and NuPAGE Bis-Tris System? Cl– (pH 6.4). — MES or MOPS (–) serves as the trailing ion in the running buffer. The running buffer ions are Tris (+), MOPS (–)/MES (–), and dodecyl sulfate (–) (pH 7.3–7.7). — Bis-Tris (+) is the common ion present in the gel, whereas Tris (+) is provided by the running buffer. The combination of lower-pH gel buffer (pH 6.4) and lower