Description

Biofargo Cell-Free Protein Synthesis Kit Max produces protein in vitro from an E. coli lysate, with the redox conditions of the system already optimised for disulfide-bond formation. Transcription and translation are coupled in one tube, so a DNA or RNA template goes in and folded protein comes out without a living host.

Expression starts within 1–2 hours and peak yield is reached in 8–24 hours, so a construct can be tested the same day it is built. Reported yields reach more than 3 mg/mL. Because nothing has to stay alive, the reaction tolerates work that kills cells — toxic proteins, unnatural amino acids, labelled precursors and direct addition of cofactors.

The kit is supplied lyophilised, which keeps it stable at −20 °C for 12 months and lets it ship on blue ice rather than dry ice. Reactions run in tubes or 96-well plates and scale linearly from microlitres upward. A pJL1-sfGFP control plasmid is included, so a failed run can be told apart from a failed construct by eye. The Max formulation needs no separate disulfide-bond enhancer: the oxidising conditions are built into the system.

For Research Use Only. Not for use in diagnostic or therapeutic procedures, or in food or pharmaceuticals.

Specifications

Parameter	Specification
Product Name	Biofargo Cell-Free Protein Synthesis Kit Max (Lyophilized)
Catalog Number	BF-P3002
Expression System	E. coli cell-free system with optimised disulfide / redox conditions
Pack Size	20 reactions (50 µL reaction scale)
Format	Lyophilized
Reported Yield	Up to >3 mg/mL
Time to Protein	Expression within 1–2 h; peak yield at 8–24 h
Template	Plasmid DNA, linear DNA, PCR product, RCA product or mRNA
Template Requirement	T7 promoter, ribosome binding site and T7 terminator; final concentration 5–10 µg/mL
Disulfide-Bond Folding	Optimised within the system; no separate enhancer required
Reaction Temperature	25–30 °C with vigorous shaking
Reaction Volume	Typically 50–100 µL; scales linearly; tubes or 96-well plates
Positive Control	pJL1-sfGFP control plasmid, 2 µg (Ex/Em 485 / 528 nm)
Storage	−20 °C, valid 12 months. After reconstitution: −20 °C up to 1 month, −80 °C up to 12 months
Shipping	Blue ice
Intended Use	For research use only

Features

Protein the same day — expression starts in 1–2 hours and peaks at 8–24 hours, against days for transformation, culture and induction.
Yields up to >3 mg/mL, several times what a standard cell-free lysate delivers.
Disulfide bonds handled in-system — the redox conditions are optimised in the formulation, so no separate disulfide-bond enhancer has to be bought or titrated.
Open reaction — nothing has to survive, so toxic proteins, unnatural amino acids, isotope labels and cofactors can be added directly.
Any common template — plasmid, linear DNA, PCR product, RCA product or mRNA, with no cloning host required.
Lyophilized format — 12 months at −20 °C and shipping on blue ice rather than dry ice.
Scales and parallelises — reactions run linearly from microlitres up, in tubes or 96-well plates.
sfGFP positive control included, so a failed reaction is distinguishable from a failed construct by eye.

Applications

Antibody formats: Express full-length antibodies, nanobodies and antibody fragments that depend on correct disulfide pairing.
Cytokines and membrane proteins: Express disulfide-rich secreted and membrane proteins that fold poorly in a reducing cell-free system.
Construct screening: Test many designs in parallel in 96-well plates before committing to a cell-based expression run.
Toxic and difficult proteins: Express proteins that kill or are not tolerated by a living host.
Protein engineering: Pair with Golden Gate or Gibson assembly and PCR or RCA templates for rapid design–build–test cycles.
Labelled protein: Add unnatural amino acids or isotope labels directly to the reaction for structural and mechanistic work.
Enzyme and assay reagents: Produce small amounts of active enzyme for assay development without a fermentation step.

Kit Components

Component	Volume	Storage
Cell-free system solution A Max (Lyophilized)	1 vial (300 µL after reconstitution)	−20 °C
Cell-free system solution B (Lyophilized)	1 vial (600 µL after reconstitution)	−20 °C
CFPS-Control Plasmid (pJL1-sfGFP)	2 µg	−20 °C

Storage & Shipping

Ship on blue ice.
Store the lyophilized kit at −20 °C. Valid for 12 months.
After reconstitution, store at −20 °C for no more than 1 month, or at −80 °C for no more than 12 months.
Keep reagents on ice while assembling reactions and avoid repeated freeze-thaw cycles.

Protocol Overview

Reconstitute solution A with 300 µL and solution B with 500 µL of nuclease-free water, and mix thoroughly.
Prepare the template. Plasmids should be column-purified; PCR products need roughly 200 bp of flanking sequence upstream of the T7 promoter and downstream of the T7 terminator to protect the linear fragment from exonucleases. Quantify the template accurately before use.
Assemble the reaction on ice in a nuclease-free tube. For a 50 µL reaction: 15 µL solution A (30%), 30 µL solution B (60%), template to a final 5–10 µg/mL, and nuclease-free water to 50 µL.
Incubate at 25–30 °C with vigorous shaking. Maximum yield is normally reached by about 8 hours; an overnight 16-hour reaction also works. Lowering the temperature to 25 °C helps some proteins fold, and the reaction time should then be extended.
For reactions above 100 µL, use a vessel with enough headspace or aeration — a shake flask at 200 rpm — since oxygen transfer limits yield at larger scale.
Take 1 µL of total reaction for total protein, or of the supernatant for soluble protein, and run SDS-PAGE.
Run the supplied pJL1-sfGFP plasmid alongside as a positive control. A successful reaction shows visible green fluorescence, and can be quantified on a plate reader at Ex/Em 485 / 528 nm.

Frequently Asked Questions (FAQs)

Common questions about Biofargo Cell-Free Protein Synthesis Kit Max (Lyophilized).

How long does a reaction take?

Expression begins within 1–2 hours. Maximum yield is normally reached by about 8 hours at 25–30 °C, and an overnight 16-hour reaction also works. Running at 25 °C helps some proteins fold but needs a longer incubation.

What yield should I expect?

The kit is specified at up to more than 3 mg/mL. Actual yield varies a great deal with the target protein, so treat the figure as the system's capability rather than a guarantee for a given construct. Run the supplied sfGFP control to confirm the system is performing before judging a difficult target.

What templates can I use?

Plasmid DNA, linear DNA, PCR products, RCA products from phi29 amplification, or mRNA. The template needs a T7 promoter, a ribosome binding site and a T7 terminator, at a final concentration of 5–10 µg/mL.

Which vectors work, and which do not?

The included pJL1-sfGFP can be used as a cloning vector, and pET-series plasmids that carry the T7 promoter without the lactose operon — such as pET-9a and pET-23a — are compatible. Plasmids containing the lac operon, pET28a among them, significantly reduce yield and are not recommended.

How should I prepare a PCR-product template?

Design the forward primer about 200 bp upstream of the T7 promoter and the reverse primer about 200 bp downstream of the T7 terminator. Those flanks protect the linear fragment from endogenous exonucleases, and the product can then be used without purification.

Does plasmid purity matter?

Yes, more than for most applications. Use a high-purity plasmid prep with a separate deproteinization wash buffer and no RNase A carryover, and elute in nuclease-free water. Plasmid that has not been column-purified cannot be used directly in the reaction.

Can it make disulfide-bonded proteins?

Yes. The Max formulation has the oxidising and redox conditions optimised within the system, so full-length antibodies, nanobodies, antibody fragments, cytokines and membrane proteins can be expressed without adding a separate disulfide-bond enhancer.

How do I scale the reaction up?

Reactions scale linearly. A typical run is 100 µL in a 2 mL microcentrifuge tube. Above that, oxygen transfer starts to limit yield, so use a vessel with sufficient headspace or aeration — a shake flask at 200 rpm.

How do I know the reaction worked?

Run the supplied pJL1-sfGFP control plasmid. A successful reaction fluoresces green and can be quantified on a plate reader at Ex/Em 485 / 528 nm. For the target protein, run 1 µL of total reaction or supernatant on SDS-PAGE.

How is it stored and shipped?

It ships on blue ice and is stored lyophilized at −20 °C, where it is valid for 12 months. Once reconstituted, keep it at −20 °C for up to 1 month or at −80 °C for up to 12 months.

Can it be used for clinical or diagnostic purposes?

No. This product is for research use only and is not intended for human, diagnostic or therapeutic use.

Related Products

Biofargo Cell-Free Protein Synthesis Kit — the standard formulation, for proteins that do not depend on disulfide bonds.

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