Sauer:Expression/purification of 35S-Met proteins/Native purification
CAUTION: Still working on this.--Kathleen 14:30, 11 Aug 2005 (EDT)
Jon's protocol
Expression
1. Inoculate 4 mL of LB+100µg/mL ampicillin with glycerol stock of JK10 (BL21 cells -SG1146A with SlyD::kan) transformed with desired plasmid. Grow over night @ 37˚C.
2. Inoculate 50mL of LB/Amp with 0.5mL of overnight culture. Shake @ 37˚C to OD600 = 1.0-1.5.
3. During the growth for step 2, prepare M9+MAM media about 1 hr before step 4:
M9 + MAM Media 50mL 1L 40% Glucose 0.5 mL 2 mg/mL Thiamine 50 µl 10 mM Trace Metals Mix 50 µl 100 mM FeCl3•6H2O 50 µl 500 mM CaCl2•2H2O 50 µl 1.0M MgCl2 50 µl MAM 1.25 mL 2xM9 25 mL H2O 23 mL
See section “Buffers and Solutions
4. Spin 50 mL culture in a 50 mL conical tube for 5 min @ 4000 rpm in the Beckman J6.
5. Discard LB supernate, and resuspend cell pellet in 50 mL M9 + MAM media.
6. Incubate 37˚C for 15-20 minutes in shaking waterbath.
7. Induce cells with 0.5 mM IPTG, incubate 15-20 minutes.
8. Add 2 mCi Expre35S35S label mix to culture (about 180µl for a 11.08 mCi/mL stock). Shake for an additional 2 hrs.
9. Harvest cells by spinning in J6 20 minutes @ 4000 rpm. Discard LB supernate.
10. Resuspend pellet in 5 mL 10 mM NTA buffer *.
11. Freeze –80˚C, thaw RT, freeze –80˚C again until ready to purify.
Purification
12. Thaw resuspended, frozen cells at RT. Make 8 x 750µl aliquots in 1.5 mL microfuge tubes.
13. Spin 15 min at 13200 rpm in benchtop centrifuge @ 4˚C.
14. During step 13, prepare NTA column: Apply 250µl of Qiagen NTA resin to a BIO-RAD poly-prep disposable column, and wash with 10 mL H2O.
15. Remove supernate from step 13, and apply to NTA column. Collect flow-through.
16. Wash column with 4 x 10 mL 10 mM NTA buffer *. Collect first 30 mL of wash separately from last 10 mL of 10 mM NTA buffer.
17. Elute material from the column with 4 x 5 mL 250 mM NTA buffer *. Collect each 5 mL elution separately.
18. Final Elute with 5mL 1.0 M NTA buffer *.
19. Test all fractions for I27 by SDS PAGE (Coomassie stain and phosphorimager detection).
20. Pool all fractions containing I27, and concentrate to 250-500µl using a Millipore centrifugal filtration device (4 or 15 mL 5 NMWL).
21. Dialyze concentrated material 2 x 4L 1xPBS.
Buffers and Solutions
10 mM Trace Metals Mix MAM (Methionine Assay Medium) 12.4 mg/mL (NH4)(Mo07)24•4H2O 105 g/L DIFCO MAM (dissolved in H2O) 2.4 mg/mL CoCl2•6H2O 2.5 mg/mL CuSO4•5H2O 2xM9 media - from kitchen, but here’s the recipe: 0.62 mg/mL H3BO3 4.4 g/L Na2HPO4•7H2O
2 mg/mL MnCl2•4H2O 6g/L KH2PO4
1.4 mg/mL ZnCl2 1g/L NaCl 2g/L NH4Cl 10 mM NTA buffer 20 mM HEPES, pH 7.6 500 mM NaCl 10 mM Imidazole (250 mM and 1.0 M NTA buffer are identical to the 10 mM NTA buffer except they have 250 mM and 1.0 M imidazole, respectively)
