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

Kathmc: /* Purification buffers */

2006-05-17T23:41:37Z

Purification buffers

←Older revision		Revision as of 23:41, 17 May 2006
Line 117:		Line 117:

	[[Sauer:Expression/purification of 35S-Met proteins]]		[[Sauer:Expression/purification of 35S-Met proteins]]
		+
	[[Protocols\| Back to Protocols]]		[[Protocols\| Back to Protocols]]

Kathmc at 23:41, 17 May 2006

2006-05-17T23:41:11Z

←Older revision		Revision as of 23:41, 17 May 2006
Line 1:		Line 1:
	{{Sauer lab sidebar}}		{{Sauer lab sidebar}}
-
-	~~'''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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	[[Sauer:Expression/purification of 35S-Met proteins]]		[[Sauer:Expression/purification of 35S-Met proteins]]
		+	[[Protocols\| Back to Protocols]]

Kathmc: I27 expression/purification moved to Sauer:Expression/purification of 35S-Met proteins/Native purification: more general title

2006-05-17T23:39:07Z

I27 expression/purification moved to Sauer:Expression/purification of 35S-Met proteins/Native purification: more general title

←Older revision

Revision as of 23:39, 17 May 2006

Kathmc at 23:37, 17 May 2006

2006-05-17T23:37:30Z

←Older revision		Revision as of 23:37, 17 May 2006
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-	~~'''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~~
-
-	~~'''remember to add the appropriate antibiotic!'''~~

	4. Pellet cells.		4. Pellet cells.
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	==Buffers and Solutions==		==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)'''		'''MAM (Methionine Assay Medium)'''
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-	'''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 102:		Line 105:
	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
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	10 mM Imidazole		10 mM Imidazole

-	(20 mM, 250 mM~~, and 1.0 M~~ NTA buffer are identical to the 10 mM NTA buffer except they have 20 mM, 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]]

Kathmc: /* Purification */

2006-05-17T23:33:49Z

Purification

←Older revision		Revision as of 23:33, 17 May 2006
Line 54:		Line 54:

	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.
		+	*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.

-	~~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 10 mM NTA.
-		+
-	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.

-	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.~~	+	16. Wash column with 50 mL 20 mM NTA buffer. Collect washes.
-		+
-	~~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)~~.	+	17. Elute material from the column with 4 x 1 mL 250 mM NTA buffer. Collect each 1 mL elution separately.

-	20. ~~Pool~~ all fractions ~~containing protein,~~ and ~~concentrate~~ to ~~250-500µl using a Millipore centrifugal filtration device (4 or 15 mL 5 NMWL)~~.	+	18. Run all fractions on an SDS PAGE gel. Use coomassie staining and phosphorimager detection/film to determine the purity of your protein..

-	21. ~~Buffer exchange by column or dialysis into desired buffer~~.	+	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==		==Buffers and Solutions==

Kathmc: /* Buffers and Solutions */

2006-05-17T23:29:22Z

Buffers and Solutions

←Older revision		Revision as of 23:29, 17 May 2006
Line 114:		Line 114:
	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, 250 mM, and 1.0 M NTA buffer are identical to the 10 mM NTA buffer except they have 20 mM, 250 mM, and 1.0 M imidazole, respectively)


	[[Sauer:Expression/purification of 35S-Met proteins]]		[[Sauer:Expression/purification of 35S-Met proteins]]

Kathmc: /* Expression */

2006-05-17T23:28:26Z

Expression

←Older revision		Revision as of 23:28, 17 May 2006
Line 31:		Line 31:
	23 mL H<sub>2</sub>O		23 mL H<sub>2</sub>O

-	~~See section “Buffers and Solutions"~~	+	'''remember to add the appropriate antibiotic!'''

		+	4. Pellet cells.

-	4. ~~Spin 50 mL culture~~ in a 50 mL ~~conical tube for 5 min @ 4000 rpm in the Beckman J6~~.	+	5. Discard LB supernatent, and resuspend cell pellet in 50 mL M9 + MAM media.

-	5. ~~Discard LB supernate, and resuspend cell pellet in 50 mL M9 + MAM media~~.	+	6. Incubate 37˚C for 20 minutes.

-	6. ~~Incubate 37˚C for 15-~~20 minutes ~~in shaking waterbath~~.	+	7. Induce cells with 1 mM IPTG, incubate 20 minutes.

-	7. ~~Induce cells with 0~~.~~5 mM IPTG, incubate 15-20 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.

-	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 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.

-	9. ~~Harvest cells by spinning~~ in ~~J6 20 minutes @ 4000 rpm. Discard LB supernate~~.	+	10. Resuspend pellet in 5 mL 10 mM NTA buffer.

-	~~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.
-		+
-	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.		*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==

Kathmc at 20:09, 17 May 2006

2006-05-17T20:09:01Z

←Older revision		Revision as of 20:09, 17 May 2006
Line 1:		Line 1:
	{{Sauer lab sidebar}}		{{Sauer lab sidebar}}
-	~~=Jon's protocol=~~

		+	'''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==

-	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:
Line 49:		Line 49:

	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.
-		+	*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.
		+

	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 *.

-	19. Test all fractions for ~~I27~~ by SDS PAGE (Coomassie stain and phosphorimager detection).	+	19. Test all fractions for protein 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).	+	20. Pool all fractions containing protein, 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~~.	+	21. Buffer exchange by column or dialysis into desired buffer.

Kathmc at 01:23, 21 September 2005

2005-09-21T01:23:53Z

←Older revision		Revision as of 01:23, 21 September 2005
Line 118:		Line 118:

	[[Sauer:Expression/purification of 35S-Met proteins]]		[[Sauer:Expression/purification of 35S-Met proteins]]
-
-	~~[[Sauer Lab\|Back to the Sauer Lab]]~~

Kathmc at 18:24, 21 August 2005

2005-08-21T18:24:07Z

←Older revision		Revision as of 18:24, 21 August 2005
Line 1:		Line 1:
-	~~'''CAUTION: Still working on this.'''--[[User:Kathmc\|Kathleen]] 14:30, 11 Aug 2005 (EDT)~~	+	{{Sauer lab sidebar}}
-		+
	=Jon's protocol=		=Jon's protocol=