Frequently Asked Questions

It is possible to order more tubes and caps directly from our supplier Sarstedt (Product number 72.730.711 for the tubes and 65.716.727 for the caps). The caps need to be perforated using a syringe needle with an appropriate gauge.

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

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.

Contact us for details of reactions at millilitre and larger volumes

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.

The pALiCE01 and pALiCE02 vectors contain Strep tags. You can therefore check the expression of your protein-of-interest by Western Blot using an anti-Strep HRP-conjugated antibody.

You can use any buffering system of your choice, provided it is in a pH range between 7.0-7.4, as this allows for optimal protein activity. 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.

Normally we recommend a centrifugation step to separate cytosolic components from the microsomes. This ensures that any possible contamination in the cytosol will not interfere with further downstream processing. However, in certain cases this centrifugation step can be skipped, and the release of proteins expressed in the microsomes be achieved by simple addition of the mild detergent DDM (n-Dodecyl-β-Maltoside) to the reaction mix. We recommend to optimize expression conditions for each protein class individually to estimate the tradeoff between yield and purity of this method of protein preparation for downstream applications.

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.

We generally recommend purifying your protein-of-interest from at least 500 µL of lysate depending on your downstream application. Magnetic beads which can bind StrepII-tagged proteins (MagStrep “type3” Strep-Tactin beads from iba Lifescience, for example) are the best options for very low-volume purifications.

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. We have also incorporated a variety of restriction enzyme recognition sites into the vector to use if you prefer restriction cloning.

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.

Our preferred method of plasmid DNA preparation is by anion exchange chromatography to obtain ultrapure DNA. Silica-based preparation methods may contain residual RNases which can affect the yield of ALiCE®.

We recommend using Gibson assembly cloning or variants thereof as it removes the need to use restriction enzymes in your cloning setup. In theory, your own vector can be used, however, be aware that pALiCE contains segments upstream and downstream of the gene-of-interest that are needed for optimal expression in ALiCE®. We can only offer the best support possible for our own vectors.

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.