|Catalog No.||Product||Quantity||SDS||Price (JPY)||Instruction|
|PFS052-0.5-EX||EF-P||500 µL for reaction||US
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“EF-P” is a supplement by adding to PUREfrex®, cell-free protein synthesis kits.
EF-P (elongation factor P) is one of translation factors in Escherichia coli (E. coli) and a homolog of eukaryotic initiation factor 5A (eIF5A). EF-P improves the synthesis of protein containing consecutive proline residues such as Pro-Pro-Pro and Pro-Pro-Gly by promoting the formation of peptide bonds.
Lysine at 34th of EF-P is post-translationally modified to β-lysllysine by EpmA (YjeA/GenX) and EpmB (YjeK) and the modification is important for the activity. “EF-P” kit includes post-translationally modified EF-P from E.coli.
- Improvement of the translation efficiency of consecutive proline residue-containing proteins by EF-P.
|EF-P||12.5 μL||EF-P with no tags (40 µM)||-80 °C*2|
|Dilution buffer||500 μL||30% glycerol buffer||-20 °C|
*1) Indicated temperature shows storage temperature after opening the kit. Please store the kit at -80°C before opening.
*2) For storage at -80°C, the rest of solution should be frozen rapidly in liquid nitrogen or dry ice/ethanol. Please divide into aliquots, if necessary, and avoid refreeze and thaw as much as possible.
This is a standard protocol for synthesizing proteins using EF-P and PUREfrex® 2.1 (#PF213). For example, please assemble 20 µL of reaction mixture as below, in which the final concentration of each reagent is 0.5 mM Cysteine, 4 mM GSH and 1 µM EF-P.
- Thaw Solution I, Cysteine and GSH by incubation at room temperature or 37 °C for 1 minute for completely dissolving, and then cool on ice.
- Thaw Solution II, III and EF-P on ice.
- Mix each solution by vortex and centrifuge briefly to collect each solution at the bottom.
- Assemble the reaction mixture in a tube as follows. Add the template DNA to 0.5 – 3 ng/µL per 1 kb.
Water 6.5-X μL Solution I *1 8 μL 10 mM Cysteine 1 μL 80 mM GSH 1 μL Solution II 1 μL Solution III 2 μL 40 µM EF-P 0.5 μL Template DNA *2 X μL Total 20 μL
- Incubate the tube at 37 °C for 2 – 6 hours with heat block or water bath.
- Analyze the synthesized products. Please add the same amount of H2O to the reaction for the sample of SDS-PAGE.
Cell-free protein Synthesis Kits
Supplements – Chaperones –
This is supplement for PUREfrex® which is molecular chaperone to be aggregate-prone protein in a soluble form.
Supplements – Forming disulfide bonds –
This is supplement for PUREfrex® to synthesis proteins containing disulfide bonds in an active form.
EF-P is developed for in vitro research only. EF-P should not be used for the therapy, diagnostic or administration to animals including human and should not be used as food or cosmetics etc. To avoid the contamination of nuclease, nuclease-free-treated water, reagents and materials should be used. We also recommend wearing gloves and mask.
“PUREfrex® is registered in U.S. Patent and Trademark Office”