Frequently Asked Questions

It is possible to order more tubes and caps, please reach out to us using the contact form.

ALiCE® is shipped on dry ice. Please contact us immediately if all the dry ice has been sublimated upon receipt of your parcel.

You can pay by either Credit/Debit Card, or by direct bank transfer. Full details can be found in our online Shop.

Proteins that require post-translational modifications should be targeted to the microsomes present in our ALiCE® reaction mix. This can be achieved by cloning your gene-of-interest into the pALiCE02 vector which contains the honeybee melittin signal peptide (MSP).

ALiCE® is a complex lysate containing our proprietary MasterMix solution for optimal expression conditions. Specific co-factors can be added and should not interfere with the transcription/translation process. Some of these co-factors might already be present in the mixture and should be taken into account when performing enzymatic assays. Contact our team for more specific details.

We have a dedicated product called ALiCE for Scale-Up which supports ALiCE reactions ranging from 5 mL to 100 mL. For reaction volumes outside of these volumes, please contact us using our contact form.

Yes, disulfide bonds are formed in the microsomes contained in our lysate. pALiCE02 contains a melittin signal peptide sequence upstream of the cloning site for your gene-of-interest. This targets your protein to the microsomes for processing.

To avoid dilution of the ALiCE® reaction mixture we recommend a concentraction of at least 200 ng/µL of plasmid DNA. The dilution factor has to be considered when comparing different DNA concentrations for optimal expression conditions. The recommended final concentration of plasmid DNA is 5 nM with a range of 2.5-20 nM for protein-specific testing purposes.

We perform extensive quality control checks on each batch of lysate and guarantee an expression level of at least 2 mg/mL for our reference protein eYFP. These checks cover parameters from a variety of different categories (biophysical, biochemical, process-specific). There is also a high degree of reproducibility for microsomally expressed proteins.

For simple proteins expressed using pALiCE01 requiring no ER/Golgi-specific post-translational modifications, protein yields of up to or above 2 mg/mL before purification can be expected. For more complex proteins or membrane proteins expressed using pALiCE02, pre-purification yields are routinely between 0.05 and 0.5 mg/mL.

ALiCE® is plant derived, so the N-glycans installed mostly consist of high-mannose type glycans, and there is no sialylation

The pALiCE01 vector can be used directly as a positive control for system function and successful reaction setup. It expresses a StrepII-tagged eYFP product that is visible by eye, can be measured with fluorescence readout (excitation 514 nM, emission 530 nM), visualized on an SDS PAGE and/or by Western blot with and anti-Strep HRP-conjugated antibody. Customer protein of interest (POI) production can be visualized by SDS PAGE and Western blot, provided an appropriate tag is introduced for antibody detection.

You can use any buffering system of your choice, provided it is in a pH range between 7.0-7.4. A buffer concentration range from 10 mM to 50 mM is recommended. Buffers at a higher concentrations may interfere with SDS-PAGE and should be avoided.

Proteins expressed in the microsomes are encapsulated by a lipid bilayer and mostly inaccessible for any analysis purposes. The mild detergent DDM (n-Dodecyl-β-Maltoside) breaks the lipid bilayer in a gentle way to release the protein-of-interest. Membrane-bound proteins (such as G-protein coupled receptors) can be accessible to assay components without requiring detergent treatment of the microsomal membrane.

Depending on the required purity for your protein assay and possible interference from lysate components with your assay evaluation, proteins can also be analyzed directly in the reaction mix. There are good examples with direct activity measurement of G-protein coupled receptors in the ALiCE® reaction mix and antibody-antigen target engagement with the antibody being expressed in the microsomes of the ALiCE® reaction mix.

The addition of 8 M Urea can maximize the recovery of protein from the microsomes, but will also result in the denaturation of your target protein. Further purification steps would be required for efficient refolding. Contact us for a more detailed explanation of this type of experiment.

Yes, proteins can be purified from 50 µl lysate reactions. We recommend using magnetic beads appropriate to your protein of interest (e.g Dynabeads™ Protein A for antibody products or MagStrep Strep-TactinXT beads for Strep-tagged proteins).

Our lysate contains His-tagged T7 polymerase. For this reason, we recommend using a StrepII-tag for cytosolic expression of your protein using pALiCE01. However, since the microsomes are a protected environment, devoid of T7 polymerase, it is possible recover protein expressed using the pALiCE02 vector usin a HIS-tag. However, additional steps are recommended to remove residual T7 polymerase. This can be achieved by pelleting the microsomes and washing the pellet without DDM before continuing with the normal protocol for microsome preparation.

It is possible to add mRNA as a template for protein expression but there are a few adjustments that need to be made first. Contact our customer support for more details.

The advantages of codon optimization have to be tested for each protein individually. If optimization is desired, Nicotiana tabacum should be selected as host organism.

Yes, pALiCE01 and pALiCE02 are high-copy-number plasmids.

The gene-of-interest can either be synthesized and inserted into pALiCE01 or pALiCE02 by third-party suppliers, or be amplified and inserted into the vector backbone yourself. For the latter, we recommend restriction-free cloning like Gibson cloning, though restriction sites are available for restriction digest-based cloning if preferred. For comprehensive guidance on preparing your template DNA, please see the Instruction Manual for Cell-Free Protein Expression with ALiCE® for Research.

For checking the correct insertion of your gene-of-interest into the multiple cloning site of pALiCE01 and 02, we recommend the following primer sequences: pALiCE-fw 5′-TCACATGTAATACGACTCACTATAGG-3′, pALiCE-rv 5′-GTACGCACCACGTGTGATTAC-3′. pALiCE-fw binds inside the T7 promoter region (similar primer sequences may be used for this region), pALiCE-rv binds to the TMV-3’UTR region.

Yes, provided the kit uses anion exchange chromatography to obtain ultrapure DNA. Silica-based preparation methods often contain residual RNases which significantly affect the yield of proteins in ALiCE® expressions.

It is possible to switch the signal peptide sequence to a sequence of your choice. However, we recommend the honeybee Melittin signal peptide (MSP) present in the pALiCE02 vector in order to offer optimal customer support if there is an issue with expression.

The ALiCE® reaction mix should be immediately transferred to -80°C. Please refrain from using liquid nitrogen to flash freeze the mix in any case. Keep freeze/thaw cycles to an absolute minimum. The control plasmids (pALiCE01 and pALiCE02) should be stored at -20°C or below.