Knight:Purification of His-tagged proteins/Denaturing: Difference between revisions

Knight:Purification of His-tagged proteins/Denaturing (view source)

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 *The lysis buffer requires many drops of 5M NaOH (tens) to bring to pH 8.0.
 *The elution buffer only needs a little (few drops) of 1M HCl to bring to pH 4.5.
+==Procedure==
+#Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
+#The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).
+#*Since I don't know what yield to expect, I arbitrarily did 50mL cultures assuming that my protein yield would be low and let it grow most of the day.  From looking at the culture, it was near but not at saturation but I didn't have time to take OD readings.
+#Harvest the cells by centrifugation at 4000 x ''g'' for 15 mins.
+#*The Qiagen protocol didn't specify a temperature so I did 4&deg;C.
+#Decant supernatant.
+#The cell pellet can be stored at -70&deg;C or processed immediately.
+#*I stored the pellet at -80&deg;C.
+#Thaw for 15 mins.
+#*I thawed at room temperature since the next step happens at room temperature.
+#Resuspend in 1mL Lysis Buffer (see above).
+#*Transferred to 2mL eppendorf tube.
+#Incubate cells with agitation for 1 hr at room temperature.
+#*Used an orbis shaker on the bench to do this temp (usually kept in 37&deg; incubator).
 ==Notes==