Difference between revisions of "Purification of GST-HA-S6K"
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| + | __NOTOC__ | ||
==Materials== | ==Materials== | ||
| − | *GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need | + | *GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need 500 ug per 10 plates of cells |
*293A Cells. | *293A Cells. | ||
*Glutathione-Sepharose | *Glutathione-Sepharose | ||
| Line 10: | Line 11: | ||
==Protocol== | ==Protocol== | ||
===Transfection of Cells=== | ===Transfection of Cells=== | ||
| − | #Split cells into 10 | + | #Split 293A cells into 10-150 mm dishes in DMEM/FBS with '''no antibiotics'''. |
| − | # | + | #Transfect when cells are 90-95% confluent. |
| − | #Combine | + | #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. | #After 5 minutes, combine the DNA solution with the Lipofectamine solution. | ||
| − | #Wait 20 min, then add | + | #Wait 20 min, then add ~2 mL to each plate of cells. |
| − | #After ~4h refeed with normal media ( | + | #After ~4h refeed with normal media (can now contain antibiotics) and incubate overnight. |
===Purification of GST-S6K1=== | ===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) | #Wash cells with D-PBS -/- twice (10 mL per plate) | ||
| − | #Add 0.5 mL [[HNTG Buffer]] | + | #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 | #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 | #Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM | ||
#Collect supernatant (save a lysate sample) into a 15 mL Falcon tube | #Collect supernatant (save a lysate sample) into a 15 mL Falcon tube | ||
#Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h. | #Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h. | ||
| + | |||
| + | [[Category: Protein Purification]] | ||
| + | [[Category: mTOR]] | ||
| + | [[Category: Kinase Assay]] | ||
Latest revision as of 19:59, 20 September 2012
Materials
- GST-HA-S6K1 plasmid. Prepare by Cesium Chloride Preparation of DNA. Need 500 ug per 10 plates of cells
- 293A Cells.
- Glutathione-Sepharose
- HNTG Buffer
- TORC1 Kinase Buffer
- Lipofectamine 2000
- OptiMEM
Protocol
Transfection of Cells
- Split 293A cells into 10-150 mm dishes in DMEM/FBS with no antibiotics.
- Transfect when cells are 90-95% confluent.
- 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 ~2 mL to each plate of cells.
- 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 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
- Collect supernatant (save a lysate sample) into a 15 mL Falcon tube
- Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h.
