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文献和实验Purification of TFIIA from E. coli
Buffer (no urea)+ 5mM DTT+ PIs with 3 changes of buffer over approximately 20 hours.Renatured protein is centrifuged for 6 minutes at 10K in SS-34 rotor. After the renaturation step,I either concentrate the protein on a Centriprep-10 concentrator (I’ve
Purification of TFIIA from E. coli
,I either concentrate the protein on a Centriprep-10 concentrator (I’ve been told that other concentrators tend to give a lower yield)and then add no salt buffer so the final salt is 50mM KCl.Or I just dialyze the entire renatured protein in the 50mM KCl buffer
Gel Mobility Shift Assay Conditions -Mg/EDTA in Gel and Buffer
-----------------------Gels:10.5 ml (20%/0.33%)acrylamide/bis acrylamide3.5 ml 10X TGOE buffer1.75 ml 50% glycerol35 microliters 0.5 M DTT20.9 ml H2O0.3 ml 10% ammonium persulfate30 microliters TEMED10X TG0E 500 ml0.25 M Tris 15.1 g Tris1.9 M glycine 71.3 g glycinepH
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