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

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=Jon's protocol=
==Expression==
==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 overnight @ 37 ˚C.
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/Amp with 0.5mL of overnight culture.  Shake @ 37˚C to OD600 = 1.0-1.5.
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:




'''For 50 mL M9 + MAM Media:'''
 
0.5 mL 40% Glucose


50 µL 2 mg/mL Thiamine
4. Pellet cells.


50 µL 10 mM Trace Metals Mix
5. Discard LB supernatent, and resuspend cell pellet in 50 mL M9 + MAM media.


50 µL 100 mM FeCl<sub>3</sub>•6H2O
6. Incubate 37˚C for 20 minutes.


50 µL 500 mM CaCl<sub>2</sub>•2H<sub>2</sub>O
7. Induce cells with 1 mM IPTG, incubate 20 minutes.


50 µL 1.0M MgCl<sub>2</sub>
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.


1.25 mL MAM
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.


25 mL 2xM9
10. Resuspend pellet in 5 mL 10 mM NTA buffer.


23 mL H<sub>2</sub>O
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.


See section “Buffers and Solutions"
==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.


4. Spin 50 mL culture in a 50 mL conical tube for 5 min @ 4000 rpm in the Beckman J6.
13. Spin 15 min at full speed in a benchtop microcentrifuge  @ 4˚C.  


5. Discard LB supernate, and resuspend cell pellet in 50 mL M9 + MAM media.
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.


6. Incubate 37˚C for 15-20 minutes in shaking waterbath.
15. Remove supernate from step 13, and apply to NTA column.  Collect flow-through.


7. Induce cells with 0.5 mM IPTG, incubate 15-20 minutes.
16. Wash column with 50 mL 20 mM NTA buffer. Collect washes.


8. Add 2 mCi Expre<sup>35</sup>S<sup>35</sup>S label mix to culture (about 180 µl for a 11.08 mCi/mL stock).  Shake for an additional 2 hrs.
17. Elute material from the column with 4 x 1 mL 250 mM NTA buffer.  Collect each 1 mL elution separately.  


9. Harvest cells by spinning in J6 20 minutes @ 4000 rpm. Discard LB supernate.
18. Run all fractions on an SDS PAGE gel. Use coomassie staining and phosphorimager detection/film to determine the purity of your protein..


10. Resuspend pellet in 5 mL 10 mM NTA buffer *.
19. Pool all elution fractions containing your protein. If necessary,  concentrate to 250-500µl using the appropriate Millipore centrifugal filtration device.


11. Freeze –80˚C, thaw RT, freeze –80˚C again until ready to purify.
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


==Purification==
50 µL 2 mg/mL Thiamine


12. Thaw resuspended, frozen cells at RT.  Make 8 x 750 µl aliquots in 1.5 mL microfuge tubes.
50 µL 10 mM Trace Metals Mix


13. Spin 15 min at 13200 rpm in benchtop centrifuge  @ 4˚C.
50 µL 100 mM FeCl<sub>3</sub>•6H2O


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 H<sub>2</sub>O.
50 µL 500 mM CaCl<sub>2</sub>•2H<sub>2</sub>O


15. Remove supernate from step 13, and apply to NTA column.  Collect flow-through.
50 µL 1.0M MgCl<sub>2</sub>


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.
1.25 mL MAM


17. Elute material from the column with 4 x 5 mL 250 mM NTA buffer *.  Collect each 5 mL elution separately.
25 mL 2xM9


18. Final Elute with 5mL 1.0 M NTA buffer *.
23 mL H<sub>2</sub>O
 
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.


appropriate antibiotic


==Buffers and Solutions==


'''MAM (Methionine Assay Medium)'''
'''MAM (Methionine Assay Medium)'''
Line 97: Line 95:


'''2xM9 media  - from kitchen, but here’s the recipe:'''
'''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
4.4 g/L  Na<sub>2</sub>HPO<sub>4</sub>•7H<sub>2</sub>O
Line 105: Line 103:
2g/L NH<sub>4</sub>Cl
2g/L NH<sub>4</sub>Cl


===Purification buffers===


'''10 mM NTA buffer'''
'''10 mM NTA buffer'''


20 mM HEPES, pH 7.6
50 mM NaH<sub>2</sub>PO<sub>4</sub>


500 mM NaCl
500 mM NaCl
Line 114: Line 113:
10 mM Imidazole
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)
(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]]
[[Sauer:Expression/purification of 35S-Met proteins]]
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Latest revision as of 16:41, 17 May 2006

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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 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)


Sauer:Expression/purification of 35S-Met proteins

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