Purification of GST-HA-S6K: Difference between revisions

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__NOTOC__

==Materials==

*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need ~~240~~ ug per 10 plates of cells

*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need 500 ug per 10 plates of cells

*293A Cells.

*Glutathione-Sepharose

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==Protocol==

===Transfection of Cells===

#Split cells into 10 ~~new~~ dishes in DMEM/FBS with '''no antibiotics'''.

#Split 293A cells into 10-150 mm dishes in DMEM/FBS with '''no antibiotics'''.

#~~Transefect with~~ 90-95% confluent

#Transfect when cells are 90-95% confluent.

#Combine ~~240~~ ug DNA with ~~1.5~~ mL OptiMEM, and ~~600~~ uL Lipofectamine 2000 with ~~1.5~~ mL OptiMEM

#Combine 500 ug DNA with 10 mL OptiMEM, and separately 700 uL Lipofectamine 2000 with 10 mL OptiMEM

#After 5 minutes, combine the DNA solution with the Lipofectamine solution.

#Wait 20 min, then add 3 mL to each plate of cells

#Wait 20 min, then add ~2 mL to each plate of cells.

#After ~4h refeed with normal media (~~containing~~ antibiotics)

#After ~4h refeed with normal media (can now contain antibiotics) and incubate overnight.

===Purification of GST-S6K1===

#Treat cells for 30min with 100 nM Wortmannin to suppress endogenous S6K phosphorylation

#Wash cells with D-PBS -/- twice (10 mL per plate)

#Add 0.5 mL [[HNTG Buffer]] ~~per~~ plate and scrape

#Add 0.5 mL [[HNTG Buffer]] to each plate and scrape

#Collect scraped cells and incubate end over end in eppendorf tube for 30 min

#Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM

@@ Line 1: / Line 1: @@
 __NOTOC__
 ==Materials==
-*GST-HA-S6K1 plasmid.  Prepare by [[Cesium Chloride Preparation of DNA]].  Need 240 ug per 10 plates of cells
+*GST-HA-S6K1 plasmid.  Prepare by [[Cesium Chloride Preparation of DNA]].  Need 500 ug per 10 plates of cells
 *293A Cells.
 *Glutathione-Sepharose
@@ Line 11: / Line 11: @@
 ==Protocol==
 ===Transfection of Cells===
-#Split cells into 10 new dishes in DMEM/FBS with '''no antibiotics'''.
+#Split 293A cells into 10-150 mm dishes in DMEM/FBS with '''no antibiotics'''.
-#Transefect with 90-95% confluent
+#Transfect when cells are 90-95% confluent.
-#Combine 240 ug DNA with 1.5 mL OptiMEM, and 600 uL Lipofectamine 2000 with 1.5 mL OptiMEM
+#Combine 500 ug DNA with 10 mL OptiMEM, and separately 700 uL Lipofectamine 2000 with 10 mL OptiMEM
 #After 5 minutes, combine the DNA solution with the Lipofectamine solution.
-#Wait 20 min, then add 3 mL to each plate of cells
+#Wait 20 min, then add ~2 mL to each plate of cells.
-#After ~4h refeed with normal media (containing antibiotics)
+#After ~4h refeed with normal media (can now contain antibiotics) and incubate overnight.
 ===Purification of GST-S6K1===
+#Treat cells for 30min with 100 nM Wortmannin to suppress endogenous S6K phosphorylation
 #Wash cells with D-PBS -/- twice (10 mL per plate)
-#Add 0.5 mL [[HNTG Buffer]] per plate and scrape
+#Add 0.5 mL [[HNTG Buffer]] to each plate and scrape
 #Collect scraped cells and incubate end over end in eppendorf tube for 30 min
 #Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM