Sauer:Expression/purification of 35S-Met proteins/Native purification

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{{Sauer lab sidebar}}
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=Jon's protocol=
 
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'''Trying to make this a bit more general. Stayed tuned for an updated version that will be linked to the main protocol page.--[[User:Kathmc|Kathleen]] 16:09, 17 May 2006 (EDT)'''
==Expression==
==Expression==
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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 overnight @ 37 ˚C.
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1. Inoculate 4 mL of LB+appropriate antibiotic with strain containing the plasmid that expresses the protein to be labeled.  Grow overnight @ 37 ˚C.
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2. Inoculate 50mL of LB/Amp with 0.5mL of overnight culture.  Shake @ 37˚C to OD600 = 1.0-1.5.
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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:
3. During the growth for step 2, prepare M9+MAM media about 1 hr before step 4:
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11. Freeze –80˚C, thaw RT, freeze –80˚C again until ready to purify.
11. Freeze –80˚C, thaw RT, freeze –80˚C again until ready to purify.
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*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.
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12. Thaw resuspended, frozen cells at RT.  Make 8 x 750 µl aliquots in 1.5 mL microfuge tubes.
12. Thaw resuspended, frozen cells at RT.  Make 8 x 750 µl aliquots in 1.5 mL microfuge tubes.
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13. Spin 15 min at 13200 rpm in benchtop centrifuge  @ 4˚C.  
13. Spin 15 min at 13200 rpm in benchtop centrifuge  @ 4˚C.  
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18. Final Elute with 5mL 1.0 M NTA buffer *.
18. Final Elute with 5mL 1.0 M NTA buffer *.
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19. Test all fractions for I27 by SDS PAGE (Coomassie stain and phosphorimager detection).
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19. Test all fractions for protein by SDS PAGE (Coomassie stain and phosphorimager detection).
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20. Pool all fractions containing I27, and concentrate to 250-500µl using a Millipore centrifugal filtration device (4 or 15 mL 5 NMWL).
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20. Pool all fractions containing protein, and concentrate to 250-500µl using a Millipore centrifugal filtration device (4 or 15 mL 5 NMWL).
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21. Dialyze concentrated material 2 x 4L 1xPBS.
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21. Buffer exchange by column or dialysis into desired buffer.

Revision as of 15:09, 17 May 2006

SAUER LAB

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Trying to make this a bit more general. Stayed tuned for an updated version that will be linked to the main protocol page.--Kathleen 16:09, 17 May 2006 (EDT)

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:


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

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 180 µl 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.

  • 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.


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 protein by SDS PAGE (Coomassie stain and phosphorimager detection).

20. Pool all fractions containing protein, and concentrate to 250-500µl using a Millipore centrifugal filtration device (4 or 15 mL 5 NMWL).

21. Buffer exchange by column or dialysis into desired buffer.


Buffers and Solutions

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, but here’s the recipe:

4.4 g/L Na2HPO4•7H2O

6g/L KH2PO4 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)


Sauer:Expression/purification of 35S-Met proteins

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