Custom Additives: Tailoring Protein Expression for Your Research Needs
Unlock the full potential of the eProtein Discovery™ system with Custom Additives.
The Custom Additives capability empowers users to customize cell-free blends with additives of their own choice. This increases the flexibility for optimizing protein expression, folding and purification, by using user-chosen additives that are better tailored for their specific protein requirements while maintaining the automation and reproducibility provided by the eProtein Discovery™ system.
Why use Custom Additives
- Flexibility: Tailor protein expression conditions to suit your unique protein targets.
- Scalability: Achieve consistent results across different scales of production.
- Optimization: Address specific challenges like protein folding, aggregation, and activity.
- Compatibility: Verified on our system for seamless integration of up to 16 user-specified additives.
Importance by Additive Category
| Category | Importance in protein science |
|---|
| Detergents and Surfactants | Solubilizing membrane proteins, preventing aggregation, and maintaining protein stability. |
| Protein Storage Buffers | Useful for preserving protein stability and activity during storage, preventing degradation and aggregation. |
| Salts & Ionic Modifiers | Adjusting ionic strength, stabilizing proteins, and influencing solubility. |
| Cofactors & Enzyme Activators | Essential cofactors or enzymatic activators for protein functionality. |
| Buffers & pH Stabilizers | Maintaining pH stability and ionic strength, critical for optimal protein folding and activity. |
| Stabilizers & Cryoprotectants | Stabilizing proteins during expression, purification, and storage. |
| Reducing Agents | Preventing oxidation, maintaining reducing environments, and stabilizing disulfide bonds. |
| Organic Solvents | Improving protein solubility, acting as cryoprotectants, or aiding in ligand binding studies |
| Chelating Agents | Binding metal ions, preventing metal-catalyzed oxidation, and assisting protein purification. |
Preparing Custom Additive Cell-Free Blends
- Prepare custom additive solutions as desired, ensuring that no component in the final formulation of the additive solution exceeds the concentration of the identical, or closely similar, compound listed in
the compatibility table.
- All custom additive solutions should be prepared and diluted in MilliQ water, unless otherwise noted.
- Use the custom additive solution prepared in step 1 to prepare a customized cell-free blend by dispensing the appropriate volume of additive into the core reagent. Custom additives can be used as single or double additives. For a reminder of how to prepare cell-free blends, please refer to the eProtein Discovery user guide.
- Once prepared, use the customized cell-free blends to prepare a transfer plate as normal, simply replacing the standard cell-free blend wells. Aspirate from the transfer plate to the same destination cartridge ports (A12-H12) as outlined in the user guide and shown in the diagram below.
- The ability to select and annotate up to 16 custom additives has been provided in the eProtein Discovery Cloud Software
- Users should proceed to input their proteins-of-interest (POIs) as per the user guide. During the ‘Experiment Design’ phase, users will ‘Select protein and construct’ as usual.
- During ‘Select cell-free blend’ phase, users now have the option to select up to 16 custom additives (called ADD01 - ADD16) from the dropdown menus under ‘Additive 1’ and ‘Additive 2’ (please see screenshot below).
- Users should also annotate within the ‘Notes’ section what the additives and their concentrations are along with any information they might find useful so that this customization information is linked to the screening run.
Chemical Compatibility Table (Max Stock Concentrations)
This chemical compatibility list is designed to guide you on the maximum concentrations compatible
with expression, purification, and droplet movement on the cartridge. Some chemicals in the list
serve as solvents for solubilizing specific chemical additives. To improve the likelihood of success and
minimize the risk of run failures, we recommend using concentrations below the stated maximums and
performance cannot be guaranteed if these maximum values are exceeded. More details are provided in
the ‘Disclaimer’ section.
Buffers and pH Modifiers
| Additive | Max Concentration |
|---|
| Tris (pH 7.5–9.0) | 50 mM |
| HEPES (pH 7.3–8.0) | 500 mM |
| Sodium phosphate (pH 7.5) | 50 mM |
| NaOH | 100 mM |
| HCl | 30 mM |
| pH < 6 or > 9 | Not compatible |
Salts and Ionic Strength Modifiers
| Additive | Max Concentration |
|---|
| NaCl | 350 mM |
| KCl | 350 mM |
| Ammonium sulfate | 350 mM |
Reducing Agents
| Additive | Max Concentration |
|---|
| DTT | 65 mM |
| TCEP | 30 mM |
| Reduced GSH | 10 mM |
Stabilizers and Cryoprotectants
| Additive | Max Concentration |
|---|
| Sucrose | 500 mM |
| Glycerol | 10% |
| PEG8000 | 2% |
| Dextran | 5% |
| BSA | 15 mg/mL |
| Betaine | 2.5 M |
Organic Solvents
| Additive | Max Concentration |
|---|
| DMSO | 10% |
| DMF | 10% |
| Ethanol | 3% |
Chelating Agents
| Additive | Max Concentration |
|---|
| EDTA | 10 mM |
| Additive | Max Concentration |
|---|
| ATP + MgCl₂ (combined) | 10 mM each |
| Hemin | 65 µM |
| Iron(II) chloride | 1 mM |
| Ascorbic acid | 5 mM |
| FMN | 130 µM |
| TPP | 10 mM |
| Cobalt chloride | 0.60 mM |
| Biopterin | 0.60 mM |
| NADPH | 10 mM |
| Ubiquinone, Cu²⁺ | Not compatible |
Protein Storage Buffers
| Component | Max Concentration |
|---|
| PBS | 1X |
| Tris | 50 mM |
| NaCl | 250 mM |
| Glycerol | 10% |
| DTT | 1 mM |
| Imidazole | 250 mM |
Detergents and Surfactants
| Additive | Max Concentration |
|---|
| LMNG | 5% |
| CHS | 0.5% |
| GDN | 1.6% |
| Digitonin | 6.6% |
| Octylglucopyranoside (OG) | 6.2 mM |
| Brij®-35, Brij®-58 | 6.6% |
| Tween®-20, Tween®-80 | 6.6% |
| Fos-choline-12 (MAPCHO®-12) | 5.4 mM |
| Fos-choline-14 (MAPCHO®-14) | 0.3 mM |
| CHAPS | 20 mM |
| LDAO | 1.2 mM |
| Sodium deoxycholate | 0.9 mM |
| Sodium cholate hydrate | 4.6 mM |
| Dodecyltrimethylammonium chloride | 1 mM |
| NDSB-195 | 500 mM |
| BIG CHAP, CTAB, DDM, Triton® X-100 | Not compatible |
Notes
- Final reaction dilution: stock solutions are diluted 13.4X (single additive) or 6.7X (double additive).
- ATP must be used with magnesium.
- Hemin and FMN must be applied consistently in all blends if chosen.
Support
For guidance, contact Nuclera Technical Support:
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