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FAQ

What is the size of the protein that can be synthesized?

We have experience synthesizing proteins from 10 kDa to 360 kDa.

It is possible to synthesize proteins below 10 kDa, but they tend to be difficult to synthesize.

If synthesis is difficult, it may be improved by synthesizing it as a fusion protein.

Will there be any post-translation modifications?

No, glycosylation does not occur.

Phosphorylation does occur, but depending on the target protein, the amino acid may or may not be phosphorylated as well as the species of origin.

Phosphorylation, acetylation, and myristoylation can be modified by introducing the necessary enzymes and substrates.

Also, methionine aminopeptidase is included, which may be used for methionine removal depending on the sequence.

Can signal peptides be cleaved?

No, the signal peptide is not cleaved.

It is recommended to exclude the signal peptide sequence before constructing the template DNA.

What is the amount of endotoxin in our wheat germ extract?

When using the WEPRO7240 series,

- In wheat extract: 150 to 300 EU/ml

- After protein synthesis and purification: 0.1 to 0.5 EU/ml

Can I dephosphorylate the synthesized protein?

Yes, it is possible.

Can I use fluorescent labeling?

Fluorescent labeling can be performed during the translation reaction using Promega's FluoroTect GreenLys in vitro Translation Labeling System.

Can be biotin labeled into synthesize protein?

And custom synthesis service could be utilized.

We have protocol and refer to our custom service

What would happen if the protein could not be synthesized at all?

If the template construction or transcription reaction has failed, the following causes are possible.

- A termination codon has been inserted near the start codon.

The mRNA is degraded.

 

In case of failure in translation reaction

- Synthesis has stopped in the middle.

(Translation has been stopped due to the influence of amino acid sequence, or translation has been stopped due to entanglement of mRNA.) 

- It has been synthesized but has self-degraded.

Can I use existing expression vectors without modification?

We recommend to use the dedicated expression vector pEU-E01 series.

Do we need to optimize the codon?

Basically, it is not necessary.

However, codon optimization may improve the amount of synthesis for genes with highly biased sequences as shown below.

- Bias in the template sequence

- Bias in the frequency of codon usage

- Bias in the secondary structure of mRNA

 

In case of proteoliposome synthesis, avoid GC rich near the 5' –turminus reagion of template.

Is there a co-ribosome binding site (RBS) or internal ribosomal entry site (IRES)?

Yes, they correspond to the E01 sequence.

Do we need to purify mRNA?

No, it is not necessary for the basic protocol.

Is there any protease inhibitor in the product?

No, it is not added.
Does it contain lipid?

Contains trace amounts of lipid derived from wheat.

Is it possible to add surfactants during the translation reaction?

Yes, surfactants can be added.

However, if the concentration becomes too high, the following possibilities may occur.

- Loss of translation activity

- Loss of activity of the target protein

Loss of the structure of the target protein

It is necessary to conduct tests at different concentration ratios to determine the optimal conditions.  

For more details, please refer to supporting data.

Can cofactors (such as metal ions) be added during the translation reaction?

They can be added. However, if the concentration becomes too high, the amount of synthesis may decrease.

Please refer to the following for the recommended concentration (final concentration) that will not cause loss of translation activity.

- [Ca ion] Calcium acetate monohydrate [Ca(CH3COO)2・H2O]: 100 uM

[Mg ion] magnesium acetate tetrahydrate [Mg(CH3COO)2・(H2O)4] : 100 uM

[Mn ion] Manganese(II) acetate tetrahydrate [Mn(CH3COO)2・(H2O)4] : 100 uM

[Cu ion] Copper(II) acetate monohydrate [Cu(CH3COO)2・H2O] : 100 uM

[Co ion] Cobalt(II) acetate tetrahydrate [Co(CH3COO)2・(H2O)4] : 10 uM

[Ni ion] Nickel (II) acetate tetrahydrate [Ni(CH3COO)2・(H2O)4] : 100 uM

[Zn ion] Zinc acetate dihydrate [Zn(CH3COO)2・(H2O)2] : 1 mM

[Cd ion]Cadmium acetate dihydrate [Cd(CH3COO)2・(H2O)2] : 100 uM

Can DMSO be added during the complementary translation reaction?

DMSO can be added. However, at concentrations of 1% (w/w) or higher, the synthesis yield will be decreased.

How much amino acid concentration is contained in the translation reaction solution?

The final concentration of each amino acid is 0.3 mM

How much DTT is contained in the translation reaction solution?

The final concentration of DTT is 4 mM.

Is EDTA contained in the translation reaction solution?

No, it isn't.

Is EGTA contained in the translation reaction solution?
No, it isn't.

Does the translation reaction solution contain DTE?

No, it does not.

Is NaCl contained in the translation reaction solution?

No. it isn't

What is the composition of the liposome?

It is a soybean-derived azolectin.

What is the particle size of the liposome?

A few hundred nm in diameter. There is no single diameter, so there is a range of particle sizes.

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