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

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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:
-
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
+
'''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
 +
 
 +
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.
4. Spin 50 mL culture in a 50 mL conical tube for 5 min @ 4000 rpm in the Beckman J6.
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7. Induce cells with 0.5 mM IPTG, incubate 15-20 minutes.
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.
+
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.
9. Harvest cells by spinning in J6 20 minutes @ 4000 rpm.  Discard LB supernate.
9. Harvest cells by spinning in J6 20 minutes @ 4000 rpm.  Discard LB supernate.
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==Purification==
==Purification==
-
12. Thaw resuspended, frozen cells at RT.  Make 8 x 750µl 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.
13. Spin 15 min at 13200 rpm in benchtop centrifuge  @ 4˚C.  
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.
+
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.
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.

Revision as of 21:36, 11 August 2005

CAUTION: Still working on this.--Kathleen 14:30, 11 Aug 2005 (EDT)

Contents

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 overnight @ 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:


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.


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 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)

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