Sauer:Expression/purification of 35S-Met proteins/Native purification: Difference between revisions
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==Expression== | |||
1. Inoculate 4 mL of LB+appropriate antibiotic with strain containing the plasmid that expresses the protein to be labeled. Grow overnight @ 37 ˚C. | |||
== | 2. Inoculate 50mL of LB/antibiotic 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: | |||
4. Pellet cells. | |||
5. Discard LB supernatent, and resuspend cell pellet in 50 mL M9 + MAM media. | |||
6. Incubate 37˚C for 20 minutes. | |||
7. Induce cells with 1 mM IPTG, incubate 20 minutes. | |||
8. Add 2 mCi Expre<sup>35</sup>S<sup>35</sup>S label mix (catalog #NEG 772 from Perkin Elmer) to culture. Shake for an additional 2 hrs. | |||
9. Harvest cells by spinning in J6 20 minutes @ 4000 rpm. Discard supernatent appropriately. | |||
*Note: You have a lot of radiation at this step and it tends to mysteriously escape from tubes onto centrifuges, even if the tube is closed tightly. Filling tubes no more than half full seems to reduce/eliminate this problem. | |||
10. Resuspend pellet in 5 mL 10 mM NTA buffer. | |||
11. Freeze at –80˚C, thaw at room temperature, freeze at –80˚C again until ready to purify. | |||
*If desired, add lysozyme (~0.3 mg/mL final concentration), incubate on ice 20 min before refreezing. This may help with the efficiency of cell lysis. | |||
==Purification== | |||
12. Thaw resuspended, frozen cells at RT. Make 8 x 750 µl aliquots in 1.5 mL microfuge tubes. | |||
*Again, it is best not to fill the tubes more than half full at this step. | |||
13. Spin 15 min at full speed in a benchtop microcentrifuge @ 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 10 mM NTA. | |||
15. Remove supernate from step 13, and apply to NTA column. Collect flow-through. | |||
16. Wash column with 50 mL 20 mM NTA buffer. Collect washes. | |||
17. Elute material from the column with 4 x 1 mL 250 mM NTA buffer. Collect each 1 mL elution separately. | |||
18. Run all fractions on an SDS PAGE gel. Use coomassie staining and phosphorimager detection/film to determine the purity of your protein.. | |||
19. Pool all elution fractions containing your protein. If necessary, concentrate to 250-500µl using the appropriate Millipore centrifugal filtration device. | |||
20. Buffer exchange by column or dialysis into desired buffer. | |||
==Buffers and Solutions== | |||
===Expression buffers and solutions=== | |||
'''For 50 mL M9 + MAM Media:''' | |||
0.5 mL 40% Glucose | |||
50 µL 2 mg/mL Thiamine | |||
50 µL 10 mM Trace Metals Mix | |||
50 µL 100 mM FeCl<sub>3</sub>•6H2O | |||
50 µL 500 mM CaCl<sub>2</sub>•2H<sub>2</sub>O | |||
50 µL 1.0M MgCl<sub>2</sub> | |||
1.25 mL MAM | |||
25 mL 2xM9 | |||
23 mL H<sub>2</sub>O | |||
appropriate antibiotic | |||
'''MAM (Methionine Assay Medium)''' | |||
105 g/L DIFCO MAM (dissolved in H<sub>2</sub>O) | |||
'''10 mM Trace Metals Mix''' | |||
12.4 mg/mL (NH<sub>4)</sub>(Mo0<sub>7</sub>)24•4H<sub>2</sub>O | |||
2.4 mg/mL CoCl<sub>2</sub>•6H<sub>2</sub>O | |||
2.5 mg/mL CuSO<sub>4</sub>•5H<sub>2</sub>O | |||
0.62 mg/mL H<sub>3</sub>BO<sub>3</sub> | |||
2 mg/mL MnCl<sub>2</sub>•4H<sub>2</sub>O | |||
1.4 mg/mL ZnCl<sub>2</sub> | |||
'''2xM9 media - from kitchen (at MIT!), but here’s the recipe:''' | |||
4.4 g/L Na<sub>2</sub>HPO<sub>4</sub>•7H<sub>2</sub>O | |||
6g/L KH<sub>2</sub>PO<sub>4</sub> | |||
1g/L NaCl | |||
2g/L NH<sub>4</sub>Cl | |||
===Purification buffers=== | |||
'''10 mM NTA buffer''' | |||
50 mM NaH<sub>2</sub>PO<sub>4</sub> | |||
500 mM NaCl | 500 mM NaCl | ||
10 mM Imidazole | 10 mM Imidazole | ||
(250 mM | |||
(20 mM and 250 mM NTA buffer are identical to the 10 mM NTA buffer except they have 20 mM and 250 mM, respectively) | |||
[[Sauer:Expression/purification of 35S-Met proteins]] | |||
[[Protocols| Back to Protocols]] | |||
Latest revision as of 16:41, 17 May 2006
Expression
1. Inoculate 4 mL of LB+appropriate antibiotic with strain containing the plasmid that expresses the protein to be labeled. Grow overnight @ 37 ˚C.
2. Inoculate 50mL of LB/antibiotic 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:
4. Pellet cells.
5. Discard LB supernatent, and resuspend cell pellet in 50 mL M9 + MAM media.
6. Incubate 37˚C for 20 minutes.
7. Induce cells with 1 mM IPTG, incubate 20 minutes.
8. Add 2 mCi Expre35S35S label mix (catalog #NEG 772 from Perkin Elmer) to culture. Shake for an additional 2 hrs.
9. Harvest cells by spinning in J6 20 minutes @ 4000 rpm. Discard supernatent appropriately.
- Note: You have a lot of radiation at this step and it tends to mysteriously escape from tubes onto centrifuges, even if the tube is closed tightly. Filling tubes no more than half full seems to reduce/eliminate this problem.
10. Resuspend pellet in 5 mL 10 mM NTA buffer.
11. Freeze at –80˚C, thaw at room temperature, freeze at –80˚C again until ready to purify.
- If desired, add lysozyme (~0.3 mg/mL final concentration), incubate on ice 20 min before refreezing. This may help with the efficiency of cell lysis.
Purification
12. Thaw resuspended, frozen cells at RT. Make 8 x 750 µl aliquots in 1.5 mL microfuge tubes.
- Again, it is best not to fill the tubes more than half full at this step.
13. Spin 15 min at full speed in a benchtop microcentrifuge @ 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 10 mM NTA.
15. Remove supernate from step 13, and apply to NTA column. Collect flow-through.
16. Wash column with 50 mL 20 mM NTA buffer. Collect washes.
17. Elute material from the column with 4 x 1 mL 250 mM NTA buffer. Collect each 1 mL elution separately.
18. Run all fractions on an SDS PAGE gel. Use coomassie staining and phosphorimager detection/film to determine the purity of your protein..
19. Pool all elution fractions containing your protein. If necessary, concentrate to 250-500µl using the appropriate Millipore centrifugal filtration device.
20. Buffer exchange by column or dialysis into desired buffer.
Buffers and Solutions
Expression buffers and solutions
For 50 mL M9 + MAM Media:
0.5 mL 40% Glucose
50 µL 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
1.25 mL MAM
25 mL 2xM9
23 mL H2O
appropriate antibiotic
MAM (Methionine Assay Medium)
105 g/L DIFCO MAM (dissolved in H2O)
10 mM Trace Metals Mix
12.4 mg/mL (NH4)(Mo07)24•4H2O
2.4 mg/mL CoCl2•6H2O
2.5 mg/mL CuSO4•5H2O
0.62 mg/mL H3BO3
2 mg/mL MnCl2•4H2O
1.4 mg/mL ZnCl2
2xM9 media - from kitchen (at MIT!), but here’s the recipe:
4.4 g/L Na2HPO4•7H2O
6g/L KH2PO4 1g/L NaCl 2g/L NH4Cl
Purification buffers
10 mM NTA buffer
50 mM NaH2PO4
500 mM NaCl
10 mM Imidazole
(20 mM and 250 mM NTA buffer are identical to the 10 mM NTA buffer except they have 20 mM and 250 mM, respectively)
