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

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'''CAUTION: Still working on this.'''--[[User:Kathmc|Kathleen]] 14:30, 11 Aug 2005 (EDT)
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{{Sauer lab sidebar}}
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=Jon's protocol=
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==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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M9 + MAM Media                    50mL  1L
 
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40% Glucose 0.5 mL
 
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2 mg/mL Thiamine 50 µl
 
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10 mM Trace Metals Mix 50 µl
 
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100 mM FeCl3•6H2O 50 µl
 
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500 mM CaCl2•2H2O 50 µl
 
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1.0M MgCl2 50 µl
 
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MAM          1.25 mL
 
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2xM9 25 mL
 
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H2O 23 mL
 
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See section “Buffers and Solutions
 
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4. Spin 50 mL culture in a 50 mL conical tube for 5 min @ 4000 rpm in the Beckman J6.
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4. Pellet cells.
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5. Discard LB supernate, and resuspend cell pellet in 50 mL M9 + MAM media.
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5. Discard LB supernatent, and resuspend cell pellet in 50 mL M9 + MAM media.
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6. Incubate 37˚C for 15-20 minutes in shaking waterbath.
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6. Incubate 37˚C for 20 minutes.
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7. Induce cells with 0.5 mM IPTG, incubate 15-20 minutes.
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7. Induce cells with 1 mM IPTG, incubate 20 minutes.
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8. Add 2 mCi Expre35S35S label mix to culture (about 180µl for a 11.08 mCi/mL stock).  Shake for an additional 2 hrs.
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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.
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9. Harvest cells by spinning in J6 20 minutes @ 4000 rpm.  Discard LB supernate.
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9. Harvest cells by spinning in J6 20 minutes @ 4000 rpm.  Discard supernatent appropriately.
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*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.
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10. Resuspend pellet in 5 mL 10 mM NTA buffer *.
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11. Freeze –80˚C, thaw RT, freeze –80˚C again until ready to purify.
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10. Resuspend pellet in 5 mL 10 mM NTA buffer.
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11. Freeze at –80˚C, thaw at room temperature, freeze at –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.
==Purification==
==Purification==
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12. Thaw resuspended, frozen cells at RT.  Make 8 x 750µl aliquots in 1.5 mL microfuge tubes.
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12. Thaw resuspended, frozen cells at RT.  Make 8 x 750 µl aliquots in 1.5 mL microfuge tubes.
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*Again, it is best not to fill the tubes more than half full at this step.
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13. Spin 15 min at 13200 rpm in benchtop centrifuge @ 4˚C.  
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13. Spin 15 min at full speed in a benchtop microcentrifuge @ 4˚C.  
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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.
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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.
15. Remove supernate from step 13, and apply to NTA column.  Collect flow-through.
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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.
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16. Wash column with 50 mL 20 mM NTA buffer.  Collect washes.
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17. Elute material from the column with 4 x 5 mL 250 mM NTA buffer *.  Collect each 5 mL elution separately.  
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17. Elute material from the column with 4 x 1 mL 250 mM NTA buffer.  Collect each 1 mL elution separately.  
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18. Final Elute with 5mL 1.0 M NTA buffer *.
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18. Run all fractions on an SDS PAGE gel. Use coomassie staining and phosphorimager detection/film to determine the purity of your protein..
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19. Test all fractions for I27 by SDS PAGE (Coomassie stain and phosphorimager detection).
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19. Pool all elution fractions containing your protein. If necessary,  concentrate to 250-500µl using the appropriate Millipore centrifugal filtration device.
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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. Buffer exchange by column or dialysis into desired buffer.
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21. Dialyze concentrated material 2 x 4L 1xPBS.
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==Buffers and Solutions==
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===Expression buffers and solutions===
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'''For 50 mL M9 + MAM Media:'''
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0.5 mL 40% Glucose
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==Buffers and Solutions==
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50 µL 2 mg/mL Thiamine
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 +
50 µL 10 mM Trace Metals Mix
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50 µL 100 mM FeCl<sub>3</sub>•6H2O
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50 µL 500 mM CaCl<sub>2</sub>•2H<sub>2</sub>O
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50 µL 1.0M MgCl<sub>2</sub>
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1.25 mL MAM
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25 mL 2xM9
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23 mL H<sub>2</sub>O
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appropriate antibiotic
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'''MAM (Methionine Assay Medium)'''
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105 g/L DIFCO MAM (dissolved in H<sub>2</sub>O)
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'''10 mM Trace Metals Mix'''
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12.4 mg/mL (NH<sub>4)</sub>(Mo0<sub>7</sub>)24•4H<sub>2</sub>O
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2.4 mg/mL CoCl<sub>2</sub>•6H<sub>2</sub>O
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2.5 mg/mL CuSO<sub>4</sub>•5H<sub>2</sub>O
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0.62 mg/mL H<sub>3</sub>BO<sub>3</sub>
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2 mg/mL MnCl<sub>2</sub>•4H<sub>2</sub>O
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1.4 mg/mL ZnCl<sub>2</sub>
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'''2xM9 media  - from kitchen (at MIT!), but here’s the recipe:'''
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4.4 g/L  Na<sub>2</sub>HPO<sub>4</sub>•7H<sub>2</sub>O
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6g/L KH<sub>2</sub>PO<sub>4</sub>
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1g/L NaCl
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2g/L NH<sub>4</sub>Cl
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===Purification buffers===
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'''10 mM NTA buffer'''
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50 mM NaH<sub>2</sub>PO<sub>4</sub>
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10 mM Trace Metals Mix                 MAM (Methionine Assay Medium)
 
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12.4 mg/mL (NH4)(Mo07)24•4H2O 105 g/L DIFCO MAM (dissolved in H2O)
 
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2.4 mg/mL CoCl2•6H2O
 
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2.5 mg/mL CuSO4•5H2O 2xM9 media  - from kitchen, but here’s the recipe:
 
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0.62 mg/mL         H3BO3             4.4 g/L  Na2HPO4•7H2O
 
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    2 mg/mL MnCl2•4H2O 6g/L   KH2PO4
 
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1.4 mg/mL ZnCl2 1g/L NaCl
 
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2g/L NH4Cl
 
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10 mM NTA buffer
 
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20 mM HEPES, pH 7.6
 
500 mM NaCl
500 mM NaCl
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10 mM Imidazole
10 mM Imidazole
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(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)
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(20 mM and 250 mM NTA buffer are identical to the 10 mM NTA buffer except they have 20 mM and 250 mM, respectively)
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[[Sauer:Expression/purification of 35S-Met proteins]]
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Contents

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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