Make Protein Purification Buffers and Media

Getting Started

You want to make Protein Mix, so you’re going to purify some proteins. Here is a list of the buffers and media you will need to prepare.

Materials and Equipment

Name	Product	Manufacturer	Part #	Price	Storage Conditions	Link
Salts (for Buffers)
Potassium Hydroxide	Potassium hydroxide - ACS reagent, ≥85%, pellets	Sigma-Aldrich	221473-1KG	$99.90	4C to 30C	[link]
Hydrochloric acid (1M)	Hydrochloric acid 1 N, Reagent Grade, Packaging=Amber Bottle, Size=500 mL	VWR	E447-500L	$72.46	4C to 30C	[link]
HEPES	HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle	Sigma-Aldrich	H3375-500G	$431	4C to 30C	[link]
Imidazole	ReagentPlus® Imidazole, Crystalline, 99%	Sigma-Aldrich	I202-500G	$120	4C to 30C	[link]
Ammonium Chloride	Ammonium chloride,ACS reagent, ≥99.5%	Sigma-Aldrich	213330-500G	$73.50	4C to 30C	[link]
Sodium Chloride	Sodium Chloride, Redi-Dri™, anhydrous, free-flowing, ACS, ≥99%	Sigma-Aldrich	746398-1KG	$133.00	4C to 30C	[link]
Potassium Chloride	Potassium Chloride, ACS reagent, 99.0-100.5%	Sigma-Aldrich	P3911-1KG	$146.00	4C to 30C	[link]
Magnesium Chloride hexahydrate	BioXtra Magnesium chloride, Hexahydrate, Powder or Crystals, ≥99.0%	Sigma-Aldrich	M2670-100G	$50.50	4C to 30C	[link]

Supplements (for Buffers)
Glycerol	Glycerol, Molecular Biology Grade, Liquid, ≥99.0%	Sigma-Aldrich	G5516-1L	$157.00	4C to 30C	[link]
Lysozyme	Lysozyme from chicken egg white, protein ≥90%, ≥40,000 units/mg protein, lyophilized powder	Sigma-Aldrich	L6876-1G	$76.90	-25C to -15C	[link]
TCEP-HCl (0.5M; presuspended)	Thermo Scientific, Bond-Breaker® Tris[2-Carboxyethyl]phosphine Neutral Solution (TCEP), Application=Water-Soluble Reducing Agent, Molecular Weight=286.65, Size=5 mL	Thermo Scientific	77720	$175.65	4C to 30C	[link]
cOmplete Protease Inhibitor	cOmplete™ Protease Inhibitor Cocktail, EDTA-Free, Tablets	Roche	11873580001	$472	1C to 4C	[link]

Media
LB	Luria Broth (Miller's LB Broth), Non-Sterile, 6.8 - 7.2, Molecular Biology Grade, suitable for regular E.coli culture, Powder	Sigma-Aldrich	L3522-1KG	$221	4C to 30C	[link]
IPTG	Isopropyl β-D-thiogalactoside (IPTG), Powder, ≥99% (TLC), ≤0.1% Dioxane	Sigma-Aldrich	I6758-1G	$89.90	-25C to -15C	[link]
Kanamycin	BioReagent Kanamycin sulfate, ≥750 ug/mg, From Streptomyces kanamyceticus, Suitable for cell culture, Suitable for plant cell culture, Powder	Sigma-Aldrich	K1377-1G	$47.70	4C to 30C	[link]

Consumables
15 mL centrifuge tubes	Corning® 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar™ Cap, Sterile, 50/Rack, 500/Case	Corning	430790	$230.44	4C to 30C	[link]
10 mL Luer lock syringes	Syringes with BD Luer-Lok® Tip, BD Medical, Syringe with Luer-Lok® Tip, Volume=10 mL	BD Industrial/Difco	550-80620-PK	$102.40	4C to 30C	[link]
0.22 um syringe filters	PES Syringe Filter, 0.22μm, 30mm, Sterile	CELLTREAT Scientific Products	229747	$84	4C to 30C	[link]
0.22 um vacuum filters	Corning® 500 mL Vacuum Filter/Storage Bottle System, 0.22 µm Pore 33.2cm² PES Membrane, Sterile, 12/Case	Corning	431097	$187.14	4C to 30C	[link]

Step 1: Prepare Stock Buffers and Media

Make (or buy) the following stock solutions. Use deionized water (e.g., milliQ).

Reagent	MW [g/mol]	Amount [g]	Final Volume [mL]	Storage	Needs pH adjustment?	Needs Sterilization?
Stock Solutions
Potassium Hydroxide (1M)	56.11	14.0	250	4C to 30C	no	no
HEPES-KOH (pH=7.6; 1M)	238.3	59.5	250	4C to 30C; dark	yes	no
Imidazole-HCl (pH=7.6; 1M)	68.08	34.0	500	4C to 30C; dark	yes	no
Ammonium Chloride (1M)	53.49	13.4	250	4C to 30C	no	no
Sodium Chloride (5M)	58.44	73.1	250	4C to 30C	no	no
Potassium Chloride (1M)	74.55	18.6	250	4C to 30C	no	no
Magnesium Chloride (1M)	203.3	20.3	100	4C to 30C	no	no
Lysozyme (30 mg/mL)	14320	1	33	-25C to -15C	no	no

Media
LB	n/a	25	1000	4C to 30C		autoclave OR filter (0.22 um)
IPTG (0.5 M)	238.3	1	4.2	-25C to -15C	no	filter (0.22 um)
Kanamycin (50 mg / mL)	484.5	2.5	50	-25C to -15C	no	filter (0.22 um)

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Notes: use hydrated salts and avoid explosions!

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Notes: adjust pH then volume.

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Notes: store HEPES and Imidazole in the dark.

Step 2: Prepare Stable Buffers and Concentrates:

Make the following buffers in advance and store at 4C:

P1 Wash Buffer - used to equilibrate all columns and wash samples. P1 Wash Buffer contains a small amount of imidazole (20 mM) to reduce nonspecific protein binding to columns.

Reagent	Final Concentration [mM]	Stock Concentration [mM]	Volume to Add [mL]
HEPES-KOH	50	1000	100
Ammonium Chloride	100	1000	200
Sodium Chloride	500	5000	200
Magnesium Chloride	10	1000	20
Imidazole-HCl	20	1000	40
TCEP	1	500	4
water (milliQ)			1436
Total			2000

P2 Elution Buffer - used to elute proteins from Ni-His columns. P2 Elution Buffer contains a large amount of imidazole (500 mM) to displace 6xHis tagged proteins from Ni-His columns.

Reagent	Final Concentration [mM]	Stock Concentration [mM]	Volume to Add [mL]
HEPES-KOH	50	1000	12.5
Ammonium Chloride	100	1000	25
Sodium Chloride	500	5000	25
Magnesium Chloride	10	1000	2.5
Imidazole-HCl	500	1000	125
water (milliQ)			59.5
TCEP	1	500	0.5
Total			250

4x P3 Concentrate - used to prepare P3 Exchange and P3 Storage buffers later

Reagent	Volume to Add [mL]	Final Concentration [mM]	Stock Concentration [mM]
4x P3 Concentrate
HEPES-KOH	100	200	1000
Potassium Chloride	200	400	1000
Magnesium Chloride	20	40	1000
water (milliQ)	180
Total	500

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Notes: P3 Buffers are compatible with PURE reactions

Step 3: Prepare Working Buffers:

Make the following buffers on the same day as use:

Lysis Buffer - used to resuspend bacterial pellets for mechanical lysis by sonication. Also chemically lyses cells with lysozyme (G. gallus). Contains protease inhibitor (cOmplete) to slow proteolysis.

Reagent	Final Concentration [mM]	Stock Concentration [mM]	Volume to Add [mL]
HEPES-KOH	50	1000	5
Ammonium Chloride	100	1000	10
Sodium Chloride	500	5000	10
Magnesium Chloride	10	1000	1
Lysozyme	0.3 mg/mL	30 mg/mL	1
TCEP	1	500	0.2
cOmplete Protease Inhibitor	1 tablet / 50 mL	n/a	2 tablets
water (milliQ)			72.8
Total			100

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Notes: Lysis Buffer is unstable at 4C

P3 Exchange Buffer - used to remove Na+ and Imidazole from proteins. Filter with a 0.22 um vacuum.

Reagent	Final Concentration [mM]
HEPES-KOH	50
Potassium Chloride	100
Magnesium Chloride	10
TCEP	1

Reagent	Volume to Add [mL]
4x P3 Concentrate	125
TCEP	1
water (milliQ)	374
Total	500

Make P3 Storage Buffer. Filter at 0.22 uM by syringe filter.

Reagent	Final Concentration [mM]
HEPES-KOH	50
Potassium Chloride	100
Magnesium Chloride	10
TCEP	1
Glycerol	30% (v/v)

Reagent	Volume to Add [mL]
4x P3 Concentrate	2.5
Glycerol	3.0
TCEP (0.5 M)	0.020
water (milliQ)	4.48
Total	10

Store all buffer concentrates at 4C until ready to use (up to one month).