Getting Started
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.
Prerequisite Protocols
Measure the concentration of your proteins:
Pierce660 Assay
Protocol
Thaw your protein stocks on ice.
Measure the concentration of each protein by
Pierce660 Assay.
Calculate how much volume of each protein stock is needed for a given final volume of Protein Mix (here: 100 uL)
# | Protein | Protein Mix Concentration [ng/uL] | Stock Concentration [ng/uL] | Volume to Add per 100 uL Protein Mix [uL] |
1 | AlaRS | 538 | ||
2 | ArgRS | 15 | ||
3 | AsnRS | 169 | ||
4 | AspRS | 62 | ||
5 | CysRS | 9 | ||
6 | GlnRS | 29 | ||
7 | GluRS | 97 | ||
8 | GlyRS | 74 | ||
9 | HisRS | 6 | ||
10 | IleRS | 308 | ||
11 | LeuRS | 31 | ||
12 | LysRS | 49 | ||
13 | MetRS | 18 | ||
14 | PheRS | 131 | ||
15 | ProRS | 77 | ||
16 | SerRS | 15 | ||
17 | ThrRS | 48 | ||
18 | TrpRS | 48 | ||
19 | TyrRS | 5 | ||
20 | ValRS | 14 | ||
21 | IF1 | 77 | ||
22 | IF2 | 308 | ||
23 | IF3 | 77 | ||
24 | EF-G | 385 | ||
25 | EF-Tu | 3846 | ||
26 | EF-Ts | 385 | ||
27 | RF1 | 77 | ||
28 | RF2 | 77 | ||
29 | RF3 | 77 | ||
30 | RRF | 77 | ||
31 | MTF | 154 | ||
32 | CK | 31 | ||
33 | MK | 23 | ||
34 | NDK | 8 | ||
35 | PPiase | 8 | ||
36 | T7RNAP | 77 | ||
Total | 7430 |
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Aliquot Protein Mix (e.g., eight (8) PCR tubes of 12.5 uL each) and store at -80C until ready for use.
Resources and References
- Papers
- Original PURE paper
- OnePot PURE
Credits
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
- Developers