sfGFP purification Reagents:
Small-Scale Culture for Screening
- Streak BL21 (DE3) containing pET28/sfGFP on plates and grow up overnight at 30 C.
- Take single colonies and inoculate 4 test tubes containing 2 mL of LB/KAN. Keep 3 more tubes just LB/KAN as blanks for OD measurements and controls.
- Shake at 200 rpm and 37C to mid-log growth phase, indicated by the OD600 nm of 0.6 to 0.7. (Referenced against LB/KAN). This takes approximately 3 hours.
- Split 2 mL cultures into two 1-mL cultures and induce one of the cultures with 1 L of 1 M IPTG. Grow the other 1-mL cultures un-induced for comparison.
- After IPTG has been added, incubate for 1 h. Small-Scale Screening
- Take 500 uL of the induced and un-induced cultures. Centrifuge the cells in 1.5 mL microcentrifuge tubes for 15 min at 13000 rpm (maximum).
- Discard the supernatant and suspend pellets in 20 uL of de-ionized water and 20 uL of gel loading buffer for SDS-PAGE.
- Boil for 5 min. and check for induction using SDS-PAGE (12.5% gel) in a side-by-side comparison of induced and un-induced cultures to ensure that sfGFP is produced.
Large Scale Cultures and Purification
In the latter part of the day set up a 50 mL overnight culture of GFP in BL21(DE3) to obtain a dense culture
- Add 50 uL of 1000x KAN to 50 mL LB
- Inoculate culture medium with GFP BL21(DE3) cultures that produced GFP the best (inoculate with un-induced matched sample)
- Shake at ~200-275 rpm and 30C
- Inoculate 0.5 or 1 L LB cultures containing 50 ug/mL LB/KAN with 5 to 15 mL of the overnight culture and grow at 30C with shaking at 200 rpm until the OD600 equals 0.6-0.7. Add IPTG to a final concentration of 1 mM and continue the growth for 3 hrs.
- Harvest the cells by centrifugation at 3,000 rpm for 30 min. and discard supernatant.
- Resuspend the pellet in Buffer A. The final volume of Buffer A should be 25 mL for a cell paste obtained from a 1 L culture.
- Freeze the sample and store overnight at -80 C
- On the following day freeze/thaw the cells 3 times to break up the cell matrix.
- Add lysozyme (10 mg/50 mL) and shake gently for 20-30 min at 25C.
- Sonicate the cell slurry at 35% power and 35% duty cycle, on ice with occasional stirring, until the solution turns viscous and then returns to a less viscous consistency (about 15 minutes).
- Centrifuge the sample at 10,000 rpm at 4C for 1 h.
- Collect the supernatant and centrifuge it at 40,000 rpm at 4C for 1 h in Ti 70 rotor. Save the supernatant for affinity column purification.
Preparation of the Ni-NTA column
Washing steps of column before sample run
- 15 mL 20% of ethanol.
- 15 mL distilled water
- 15 mL freshly prepared 0.1 M NiSO4
- 15 mL of distilled water
- 15 mL of Buffer A
- Load the column with sample and adjust the rate at 30/25 mL sample using peristaltic pump or 1 mL/min in HPLC.
- Wash the column using 3 bed volumes of Buffer B or until the UV280 signal returns to baseline.
- Elute histidine tagged protein with Buffer C.
- After using the column, wash it with 5 to 10 column volumes of 0.05 M EDTA, distilled water and 20 %ethanol respectively (Column regeneration).
- Transfer the sample eluted from the Ni-NTA column using Buffer C to dialysis tubing with a 10,000 MWCO.
- Dialyze the sample using about 800 mL of Buffer A 3 times. The first round of dialysis is carried out overnight; the 2nd and 3rd rounds are carried out for 2-3 h periods.
- Assess purity of sample using SDS–PAGE (12.5% acrylamide gel).
- Determine concentration by absorbance at 485 nm
Extinction coefficient = 8.33 x 10^4 M-1cm-1 (ref. 1).
- Flash Freeze in LN2 and store @ -80C.
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