We’re here when you need us. Please get in touch if you have specific questions about your project. In the meantime, here are the answers to a few questions that you might have:
Do you recommend assay conditions for using Bactosomes
Details of our QC assays and, in some cases, assays using alternative substrates are available online for all of the recombinant enzymes that we make. Look under the individual product page for the link. We have also presented a poster looking at the effect of using different buffers on CYP3A4 activity.
Can Bactosomes be used with any predictive / modelling software?
We know from positive customer feedback that Cypex Bactosomes can be used in conjunction with the Simcyp simulator.
Do I have to change my assays significantly to use Bactosomes?
No, Bactosomes are compatible with your assay systems, you just have to take into account the change in specific enzyme activity. We recommend that some optimisation be undertaken when switching to using Bactosomes.
Are there any problems associated with the fact that Cypex cytochrome P450s are produced in E. coli?
No, there is no evidence to suggest that using a prokaryotic expression system affects the activity of the recombinant P450 in any way. The novel system used by Cypex allows the production of native human P450, a significant improvement over other bacterial expression systems using N-terminally modified cytochrome P450s.
Can I refreeze Bactosomes?
We recommend thawing and then aliquotting Bactosomes into amounts sufficient for a single use to minimise freeze thaw cycles. That said, Bactosomes will tolerate a limited number of freeze-thaw cycles, provided that care is taken and they are thawed on ice and refrozen as soon as possible after thawing. We have produced data showing the stability of Bactosomes with repeated freeze / thawing.
Can I dilute Bactosomes before aliquotting and refreezing?
We recommend storing Bactosomes with as high a protein concentration as possible, however, we have diluted Bactosomes in storage buffer (50 mM Tris-acetate pH 7.6, 250 mM sucrose, 0.25 mM EDTA) and refrozen them and found no difference in their activity after storage at -80°C. Problems have been encountered when the Bactosomes have been diluted in buffers other than the storage buffer specified.
How do I ensure the Bactosome suspension is homogeneous before use?
The best way to mix the Bactosomes suspension is to pipette it up and down gently a few times, being careful not to introduce air bubbles. Gently flicking the tube also works. We do not recommend that the tube is vortexed, we have found that the activity decreases slightly with vortexing.
What is the difference between R, HR and LR Bactosomes?
The activity of many cytochrome P450s can vary significantly with the amount of NADPH P450 reductase available. R and HR Bactosomes have high levels of NADPH P450 reductase and have, therefore, a high activity. This limits the linearity of substrate turnover with time. LR Bactosomes contain lower levels of NADPH P450 reductase than their R counterparts. These Bactosomes, while they have a lower Vmax for a given substrate, show enhanced linearity of substrate turnover with time . For a good illustration of this compare the product sheets for CYP3A4R and CYP3A4LR. The product that you choose will depend on your assay requirements.
How much can I dilute Bactosomes in my assay?
The QC for all batches of Bactosomes incorporates an assay for linearity of substrate turnover with P450. The upper limit for this is given in the accompanying Data sheet. Graphs showing typical values for the lower end of this assay are available on the product sheets on our website and in our catalogues. We have found no artefacts associated with the dilution of Bactosomes in assays however, we recommend diluting the Bactosomes at the last minute before adding them to your assay as the diluted enzyme (particularly dilutions greater than 1/10) is less stable in the absence of substrate.
Are Bactosomes available in alternative aliquot sizes / P450 concentrations?
We manufacture our off the shelf products to have the highest specific P450 content possible (pmol P450/mg protein) to allow you the greatest flexibility when diluting the products for use in your assays. We are happy to aliquot our products specifically to fit your requirements free of charge subject to a minimum order size.