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Detailed Methods for GST-Seu Purification
1) Starter cultures (~3 mL each) of GST-SEU (ampicillin and chloramphenicol were in
the media) were grown overnight at 37°C in a shaker.
2) The next morning, flasks (containing 50 mL LB + 1.25 mL 20% glucose + 40 L
(100mg/ml)ampicillin + 50L chloramphenicol(34mg/ml)) were each inoculated with 1.5
mL of starter culture. The flasks were incubated at 37°C in a shaker.
3) After ~2 hours 25 min., 4.5 mL of culture from one of the flasks was pelleted in a
microcentrifuge tube and saved.
4) 200 L of 100 mM IPTG stock was added to each flask. The flasks were incubated
again at 37°C in a shaker.
5) After ~3 hours 20 min., 2 samples of culture (3 mL each) were pelleted in separate
microcentrifuge tubes (14,000 rpm, 1 min., 4°C). The remaining culture was pelleted in
2 large centrifuge tubes (7,000 rpm, 15 min., 4°C).
6) For the small pellets in the microcentrifuge tubes (that were used to check the yield
before processing the large pellets), the following was done:
- 400 L BugBuster and 2 L protease inhibitor cocktail were added to each
microcentrifuge tube
- the pellets were resuspended by vortexing
- incubated the tubes at RT for ~30 min. on a rocker
- the tubes were spun (14,000 rpm, ~10 min., 4°C); for the remaining steps, all
samples were kept on ice
- the supernatants were transferred to new microcentrifuge tubes
- to wash the pellets: each pellet was resuspended in 1 mL of 1X Wash
Buffer(PBS+0.1% tritonX-100) by
vortexing (and by crushing chunks with a pipette tip and by pipetting up and down?);
the tubes were spun (14,000 rpm, ~2 min., 4°C); the supernatants were discarded
- the pellets were washed again
7) For the large pellets, the following was done:
- 5 mL BugBuster was added to each pellet
- 200 L PMSF and 40L protease inhibitor cocktail was added to each tube
- incubated the tubes at RT for ~2 hours on a rocker
- spun the tubes (8,000 rpm, 10 min., 4°C); discarded the supernatants
- to wash the pellets (kept the samples on ice?): 5 mL Novagen Wash Buffer
(prepared by diluting the 10X stock to 1X with autoclaved dH2O) was used to
resuspend each pellet --- it was important that no visible chunks of precipitate
remained (this was done by thoroughly crushing any chunks with a pipette tip and
by pipetting the mixture up and down); spun the tubes (8,000 rpm, 10 min., 4°C);
discarded the supernatants
-
repeated the washing steps twice. washing is done until pellet size doesn’t
decrease any further.
- 4 mL of solubilization mix (1% sarcosyl in PBS+5mm DTT) was added to each pellet
- the tubes were incubated at RT on a rocker for 20 min.
- spun the tubes (8,000 rpm, 10 min., 4°C)
- transferred the supernatants to fresh tubes; discarded the pellets (which contained
insoluble proteins)
- added Triton-X 100 to the supernatants to a final concentration of 1%? (this step
released the proteins from the micelles)
- dialyzed with PBS+0.1mm DTT at 4C O/N.
- next day the supernatants were dialyzed with PBS at 4C for three hours.
- At this stage the GST-SEU is almost pure yielding single band in SDS-PAGE gel.
- This can be further purified by binding to GST column after dialysis.