2021-08-10求助! 求用EGS与纯化蛋白交联的实验操作步骤

Cross-linking assaysThe cross-linker ethylene glycolbis(succinimidylsuccinate) (EGS) was dissolved in dimethyl sulfoxide at a concentration of 25mM. Cross-linking reactions were carried out in a reaction mixture containing 50mM Hepes pH7.5, 100mM NaCl and 25μm of the corresponding protein. As controls of dimeric and monomeric proteins, glutathione S-transferase (GST) and E. coli ferredoxin were used respectively. EGS was added to the reaction mixture at different molar proportions: 10, 20, 30 and 50-fold molar excess. The reactions were incubated for 30min at room temperature. Then, the EGS was quenched by the addition of 50mM Tris pH7.5. Samples were subjected to 12% SDS-PAGE as described elsewhere.http://jpsionline.com/admin/php/uploads/18_pdf.pdfhttp://pubs.acs.org/doi/abs/10.1021/acs.analchem.6b00281http://groups.molbiosci.northwestern.edu/morimoto/research/Protocols/III.%20Proteins/D.%20Protein-Protein/1.%20Glut.%20X-Linking.pdf