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Recombinant Catalytic Subunit Prep Protocol
Recombinant Catalytic Subunit Preparation
(cAMP-dependent Protein Kinase A)

Susan Taylor (University of California at San Diego) January 3, 1997

Clone Culture, Expression & Harvest

 1. Prepare 12 liters of sterilized YT Media by placing 18 YT  	capsules/Liter of MQ H20 and Autoclaving.  Let the media cool  	to at least 37°C and add to each flask 1 ml of a 100 mg/ml MQ  	H20 filtered ampicillin solution (1.2 g Ampicillin/12 ml H2O).     2. Add 1.1 ml of sterile YT to the frozen bacterial cell stock.   	Vortex well. Using sterile techniques, add 100 ml of this  	solution to each flask. If you inoculate the media that is at  	room temperature, let the cells grow for 8-8.5 hours  	at 37°C until OD600 is greater than 0.55 and less than 0.80. If  	you inoculate media that is warm, the cells will grow to the  	appropriate OD in less time.  **Alternate Method:  Inoculate cold media flasks (with  	Ampicillin) at night. Place flasks in the shaker and set the  	temperature to 37°C and RPM to 225. Set timer to start in the  	early morning, ex: 1:00 or 2:00 am. Let grow 8-8.5 hours at  	37oC until OD600 is approximately between 0.55-0.80. Record the  	OD in your notes.  3. When cultures reach an OD600 of 0.55 to 0.80, induce them with  	0.5 M IPTG (1.62 g/12 ml). Add 1 ml to each liter observing  	sterile technique. Final concentration of IPTG ==> 0.5 mM.   	Adjust the temperature control of the shaker to 24°C. Leave lid  	open or the temperature will continue to rise past 24°C. Grow  	for an additional 6 hours.  4. Harvest the cells by spinning in 1 liter bottles for 11 minutes  	at 5,000  RPM. Use Sorvall centrifuge.   5. Consolidate the pellets in a 40 ml centrifuge tube. Label  	completely and store the cells in the -70°C freezer.   

A Day Before Your Prep Starts or Early the First Day of Your Prep:
 6. Prepare P-11 phosphocellulose resin 	A. Stir 250 mls of 0.5 M NaOH into 12-13 g of P-11 and let sit  		for 5 minutes 	B. Filter off the supernatant and wash with MQ water (about 2  		liters) until the pH is below 11 (Use pH paper to measure  		approximate pH's) 	C. In filter funnel mix resin with 250 mls 0.5 M HCl and let  		sit for 5 minutes 	D. Filter off the supernatant and wash with MQ water (about 2  		liters) until the pH is above 3.0 	E. Transfer the P-11 into approximately 3 volumes (300 mls) of  		10X P-11 running buffer 	F. Titrate the slurry with NaOH until the pH is 6.5. Let the  		solution sit in the cold room 	G. Decant the supernatant and add 250 ml of 1X running buffer.   		Stir the slurry and pH again to 6.5 	H. Leave in cold room overnight or until you add the total cell  		supernatant. 

Day 1 of Cat Protein Prep

 1. Remove frozen pellets from -70°C freezer and let thaw at room  	temperature on bench or in a beaker of water.  Resuspend the  	thawed pellets in 240 mls 1X P-11 lysis buffer [Dilute  	10X P-11 (cold room) and add 43 ml b-me to 5mM]  2. Lyse the cells in the French Press 2X. Save 20 ml of the total  	cell lysate and record the total volume  3. Spin the lysate for 40 minutes at 15,000 RPM (JA-20 rotor) in  	50 ml centrifuge tubes. Collect and combine the supernatants.   	Take a 20 ml aliquot and record total volume. Save the pellet  	from one of the centrifuge tubes and resuspend with water 	in  	the same volume as total cell lysate. Take a 20 ml aliquot and  	save.    4. Measure and record the initial conductivity of the supernatant.  	Dilute the supernatant with cold water (4°C) until the  	 	conductivity reaches 1.2 mS/cm. Check the pH and make sure it  	is at 6.5.  Save a 50 ml aliquot of the diluted supernatant and  	record volume.  5. Check pH of the resin (6.5). Decant off supernatant of resin.   	Add resin to the diluted cell supernatant. Mix Slowly (Batch  	bind) for 3 hours minimum (or overnight) in an appropriately  	sized beaker.  

End of Day 1 or Day 2 of Cat Protein Prep

 6. Decant resin through funnel (plastic). Resuspend in 1X Running  	buffer.  7. Pour Slurry into a 100 ml column. Pack and wash with 200 mls  	of 1X running buffer (or batch wash the resin in a  	beaker for 10 minutes at 4oC.).  8. Column wash with 200 mls of: 1X Running buffer + 50 mM KPO4.   	Collect elution in bottle or beaker and save. (or batch  	wash in beaker for 10 minutes).  9. Wash with 200 mls of:  1X Running buffer + 90 mM KPO4. Collect  	elution in bottle or beaker and save. (or batch wash in  	beaker for 10 minutes and pour column).  10. Elute with 250 mls of 1X Running buffer + 250 mM KPO4.   	Collect 8-10 ml fractions.    11. Run 12.5% SDS PAGE gels. Include standard, total cell lysate  	(2 ml), lysate pellet (2 ml), lysate supernatant (2 ml),  	diluted lysate supernatant (5 ml), flow through (5 ml), 1X  	Running Buffer Wash, (20 ml), 50 mM Wash (20 ml), 90mM Wash  	(20 ml), and selected fractions (20 ml).  12. Pool wanted protein (proteins that show little or no  	contaminating bands) in a small beaker. Dialyze the  	protein in 2L of Buffer A (20 mM KPO4), pH 6.5 overnight.  

Day 2 or 3 of Cat Protein Prep

 1. Check pH of Dialysis buffer, make sure it is at pH 6.5.  2. Measure the concentration of your dialyzed protein.  Record  	concentration and volume of your pooled protein.  Take a 20 ml  	aliquot.  3. Load up to 50-60 mgs of protein onto Mono S 10/10 HR of FPLC.  4. Watch for peaks. Protein usually starts eluting between 15-25%  	of Buffer B.  5. Run a 12.5% SDS PAGE Gel.  Include standard, load (10 ml), and  	all necessary fractions  6. Run IEF Gel to determine peaks of individual fractions (if  	necessary).  7. Check Cat activity and record.  8. Dialyze protein for crystallographers in : 		50 mM Bicine 		150 mM Ammonium Acetate 		10 mM b-me          		pH 8.0  9. Dialyze protein to be frozen at -20°C in: 		100 mM MOPS 		150 mM KCl 		0.1 mM DTT            		pH 7.1  10. Record total amount of protein recovered, separating them by  	peaks.  11. Record distribution of protein and amount distributed. 

Buffers for Recombinant Cat Preparation

  Lysis buffer: 	1X			10X 		30 mM MES		300 mM MES 		1 mM EDTA		10 mM EDTA 		50 mM KCl		500 mM KCl 		5 mM b-me		 		pH 6.5			pH 6.5  P-11 Running buffer:	 			1X			10X 			30 mM MES		300 mM MES 			1 mM EDTA		10 mM EDTA 			5 mM b-me		 			pH 6.5	 		pH 6.5  Mono S: 			Buffer A:		Buffer B: 			20 mM KPO4 		20 mM KPO4 						1M KCl 			5 mM b-me or		5 mM b-me or 			  2 mM DTT		  2 mM DTT 			pH 6.5	     		pH 6.5   S-100:			1X:			10X: 			200 mM KCl		2 M KCl 			25 mM KPO4		250 mM KPO4 			5 mM b-ME 			pH 7.0			pH 7.0  For all solutions: pH when cold, add b-me to final solutions only  (350 ul/1L).