<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Getting Started

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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.

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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					 | $72.46 | 4C to 30C | [[link](<https://us.vwr.com/store/catalog/static_catalog.jsp?catalog_number=97064-756>)] |

| 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] |

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 1: Prepare Stock Buffers and Media

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• [ ] Make or buy the following stock solutions. Use Ultrapure water (18.2 MOhm, e.g., Milli-Q).
• Notes: use hydrated salts when able to and avoid explosions!
• Notes: adjust pH then volume.
• Notes: store HEPES and Imidazole in the dark.

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 2: Prepare Stable Buffers and Concentrates:

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Make the following buffers in advance and store at 4C. Add all components except TCEP, which you MUST add fresh the day of use.

• [ ] P1 Wash Buffer - used to equilibrate columns and wash samples. P1 Wash Buffer contains a small amount of Imidazole (20 mM) to reduce nonspecific protein binding to columns, which improves the purity of your protein prep.

  Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
  HEPES-KOH (pH 7.6)	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
  Ultrapure water			1436
  Total			2000

• [ ] P2 Elution Buffer - used to elute proteins from Ni columns by introducing a large amount of Imidazole (500 mM) that competes with polyhistidine purification tags to bind the column.

  Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
  HEPES-KOH (pH 7.6)	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
  TCEP	1	500	0.5
  Ultrapure water			59.5
  Total			250

• [ ] 4x P3 Concentrate - used to prepare P3 Exchange and P3 Storage Supplement later.

  Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
  HEPES-KOH (pH 7.6)	200	1000	100
  Potassium Chloride	400	1000	200
  Magnesium Chloride	40	1000	20
  Ultrapure water			180
  Total			500

  • Notes: P3 Buffers are compatible with PURE reactions

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 3: Prepare Working Buffers:

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• [ ] Make the following buffers on the same day as use:

  • [ ] Lysis Buffer - used to resuspend bacterial pellets for lysis. Also enzymatically 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 (pH 7.6)	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
    cOmplete Protease Inhibitor	1 tablet / 50 mL	n/a	2 tablets
    TCEP	1	500	0.2
    Ultrapure water			72.8
    Total			100

    • 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 filter.

  • [ ] P3 Storage Supplement - added to samples in P3 Exchange Buffer as a cryoprotectant for storage at -80C. Filter P3 Storage Supplement with a 0.22 um syringe filter. To use, add an equal volume of P3 Storage Supplement to samples in P3 Exchange Buffer (1:1). P3 Exchange Buffer supplemented with P3 Storage Supplement is called “P3 Storage Buffer”.

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

<aside> <img src="/icons/book_gray.svg" alt="/icons/book_gray.svg" width="40px" /> Resources and References

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• Papers

<aside> <img src="/icons/megaphone_gray.svg" alt="/icons/megaphone_gray.svg" width="40px" /> Credits

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Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)