<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 PURE. You’ve got the thirty-six (36) purified proteins that make up the protein component of PURE and you want to assemble Protein Mix from them. This protocol tells you how to assemble Protein Mix from individually purified proteins.

<aside> <img src="/icons/merge_gray.svg" alt="/icons/merge_gray.svg" width="40px" /> Prerequisite Protocols

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• [ ] Purify PURE proteins: Grow and Induce Expression Strains, Lyse Bacteria by Sonication, Purify Proteins by Ni2+ Gravity Column, Exchange Buffers and Concentrate by Spin Filtration
• [ ] Measure the concentration of your proteins: Pierce660 Assay

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

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• [ ] Thaw your protein stocks on ice.

• [ ] Measure your protein stock concentrations by Pierce660 Assay.

• [ ] Read the following Google Sheet template and follow its directions to assemble your Protein Mix. https://docs.google.com/spreadsheets/d/1OLFNVuiL5-f6FAD-eY3rEHRrYya3oPlh2JK1UhwY7qs/edit?usp=sharing

  https://docs.google.com/spreadsheets/d/1OLFNVuiL5-f6FAD-eY3rEHRrYya3oPlh2JK1UhwY7qs/edit?usp=sharing

  • [ ] Input the following information into blue colored cells:
    • [ ] Desired number of 10 uL PURE reactions
    • [ ] Protein stock concentrations
    • [ ] Protein stock volumes
  • [ ] Check the “Checklist” section to see if any cells are highlighted yellow. If so, follow the directions next to the highlighted cell.
  • Notes: if your Protein Mix is dilute, you can concentrate it

• [ ] Concentrate Protein Mix to the Target Final Volume (~ 12.25 mg / mL) using a 3kDa Amicon Centrifugal Filter.

  • Notes: wash and concentrate your Protein Mix after assembly

• [ ] Measure the total protein concentration of your Protein Mix:

  • [ ] Create 10x dilutions of Protein Mix (1 uL in 9 uL Ultrapure water) in triplicate.
  • [ ] Perform a Pierce660 Assay on your diluted Protein Mix samples.
  • [ ] Calculate the total protein concentration of your diluted Protein Mix samples. Average the values of each triplicate and multiply by 10 to estimate the total protein concentration of your Protein Mix.

• [ ] Dilute your Protein Mix to 12.25 mg / mL in P3 Storage Buffer (30% glycerol). If the concentration of your Protein Mix is less than 12.25 mg / mL, repeat the last two steps (concentrate your mix, then measure using Pierce660)

• [ ] Aliquot your Protein Mix (12 uL + 1 uL of head room per tube) into PCR tubes or microcentrifuge tubes and store -80C.

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

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• Papers

  • Original PURE paper

  Cell-free translation reconstituted with purified components

  • OnePot PURE

  A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

  OnePot PURE Cell-Free System | Text Page

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Credits

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

• Developers
  • Murray Lab
  • Freemont Lab
  • Shimizu lab