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Getting Started

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Ribosomes are large complexes of RNA and proteins (MW ~2.7 MDa) that are the sites of protein synthesis. Ribosomes coordinate the decoding of mRNA transcripts by tRNA and catalyze the formation of each peptide bond in new proteins, making ribosomes a key component of any protein synthesis system. Ribosomes can be purified from E. coli biomass by a variety of methods (e.g., His-tagged ribosomes can be purified by Ni-His chromatography, as in Purify Proteins by Ni2+ Gravity Column), but we recommend a two step process: (1) initial, tag-free purification by hydrophobic interaction chromatography (HIC) and (2) size-selective precipitation by ultracentrifugation. This protocol will show you how to grow E. coli A19 biomass and purify ribosomes by HIC followed by ultracentrifugation.

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Materials and Equipment

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‼️All reagents and materials must be prepared RNase-free. Use RNaseZap or 10% bleach to decontaminate plastic and glassware and rinse with nuclease-free water. We find ultrapure water (18.2 MOhm) is often sufficient for RNase-free work.

• Table 1: Reagents and Consumables
• Table 2: Equipment
• Table 3: Stock buffers

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol

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• [ ] Prepare Buffers and Media

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

  Buffer Template: https://docs.google.com/spreadsheets/d/147qsJ1dcdYtmi5C8nt7JbuJrqMnB_ckhk1la_LU6xIw/edit?usp=sharing

  • [ ] Ribosome Lysis Buffer

    Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
    HEPES-KOH (1 M, pH 7.6)	10	1000	2.5
    Magnesium Acetate (1 M)	10	1000	2.5
    Potassium Chloride (1 M)	50	1000	2.5
    TCEP-HCl (0.5M)	1	500	0.5
    Ultrapure water	-	-	242
    Total			250

  • [ ] Salting Out Buffer

    Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
    HEPES-KOH (1 M, pH 7.6)	10	1000	1
    Magnesium Acetate (1 M)	10	1000	1
    Potassium Chloride (1 M)	50	1000	1
    Ammonium Sulfate	3000	n/a	39.6 g
    TCEP-HCl (0.5M)	1	500	0.2
    Ultrapure water	-	-	92.8
    Total			100

  • [ ] Ribosome Wash Buffer

    Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
    HEPES-KOH (1 M, pH 7.6)	20	1000	10
    Magnesium Acetate (1 M)	10	1000	5
    Ammonium Sulfate	1500	n/a	99.1 g
    TCEP-HCl (0.5M)	1	500	1
    Ultrapure water	-	-	484
    Total			500

  • [ ] Ribosome Elution Buffer

    Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
    HEPES-KOH (1 M, pH 7.6)	20	1000	6
    Magnesium Acetate (1 M)	10	1000	3
    TCEP-HCl (0.5M)	1	500	0.6
    Ultrapure water	-	-	290.4
    Total			300

  • [ ] Cushion Buffer

    Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
    HEPES-KOH (1 M, pH 7.6)	20	1000	5
    Magnesium Acetate (1 M)	10	1000	2.5
    Ammonium Chloride (1 M)	30	1000	7.5
    Sucrose	30% (w/v)	n/a	75 g
    TCEP-HCl (0.5M)	1	500	0.5
    Ultrapure water	-	-	234.5
    Total			250

  • [ ] Ribosome Buffer

    Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
    HEPES-KOH (1 M, pH 7.6)	20	1000	5
    Magnesium Acetate (1 M)	6	1000	1.5
    Potassium Chloride (1 M)	30	1000	7.5
    TCEP-HCl (0.5M)	1	500	0.5
    Ultrapure water	-	-	235.5
    Total			250

  • [ ] AcOH (0.1 M)

    Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
    Acetic Acid (glacial; 17.4M)	100	17400	1.44
    Ultrapure water	-	-	248.5
    Total			250

  • [ ] EtOH (20% v/v)

    Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
    Ethanol	20% v/v	100% v/v	50
    Ultrapure water	-	-	200
    Total			250

• [ ] Cell culture:

  • [ ] Prepare overnight cultures.
    • Notes - We purify Ribosomes from A19
    • [ ] Add 5 mL Luria Broth (LB) under sterile conditions to two (2) 14 mL culture tubes.
    • [ ] Label one tube “(+)”. Add 10 uL of A19 glycerol stock to (+).
    • Notes - you can work from glycerol stocks OR colonies.
    • [ ] Label the other tube “(-)”. This will be your negative control, testing if your technique is sterile.
    • [ ] Incubate both tubes overnight shaking at 37C / (225-250) rpm / (12-16) hr.
  • [ ] Perform bulk outgrowth.
  • [ ] Pellet, wash, and store cells.

• [ ] Cell lysis:

• [ ] FPLC purification

• [ ] Ultracentrifugation

• [ ] Quality Control and Storage

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

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• Papers
• Cytiva HiTrap Butyl HP Instructions

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

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