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Protein Concentration & Buffer Exchange Guidelines

The Nuclera wash and elution buffers contain a nonionic detergent (12.5 kDa) required for effective cartridge droplet operations (Table 1). This is typically inert and well tolerated by proteins, but due to its unusually high molecular weight, can co-concentrate with proteins when using centrifugal concentrator devices, and care should be taken to use an appropriate filter MWCO to avoid problems associated with increasing the concentration of the detergent and the effect that could have on your protein of interest.

Elution Buffer formulation at pH 8.0Wash Buffer formulation at pH 8.0
  • 0.1 M Tris-Cl
  • 0.15 M NaCl
  • 50 mM biotin
  • 0.05% nonionic detergent
  • 0.1 M Tris-Cl
  • 0.15 M NaCl
  • 0.05% nonionic detergent
Table 1: Formulations of the Elution and the Wash buffers supplied in the Scale-up kit.

Detergents of this size (12.5 kDa) will be retained by filters with molecular weight cut-offs (MWCO) below 30 kDa, leading to unintended co-concentration with your protein. To prevent detergent retention and ensure optimal protein recovery, please follow these guidelines:

✅ Recommended Practice

  • For proteins > 60 kDa: Use a 30 kDa MWCO concentrator. This enables efficient separation of protein from detergents and minimizes loss of the target protein.

    • To reduce detergent content, dilute the eluted protein in a detergent-free elution buffer (Table 2) and reconcentrate using the same filter.
    • Note: Detergent removal may affect protein conformation, solubility, or alignment with screen predictions.

  • For proteins < 60 kDa: Use a smaller MWCO filter and substitute purification buffers (wash and elution, Table 2) with your own detergent-free buffer with pH maintained between pH 7-8.

    • Be aware that buffer changes may affect protein folding and yield relative to cartridge predictions.

If you have any questions, please contact Nuclera Technical Support.

Elution Buffer formulation at pH 8.0Wash Buffer formulation at pH 8.0
  • 0.1 M Tris-Cl
  • 0.15 M NaCl
  • 50 mM biotin
  • 0.1 M Tris-Cl
  • 0.15 M NaCl
Table 1: Formulations of the Elution and the Wash buffers supplied in the Scale-up kit. Table 2: Formulations of the detergent-free Elution and the Wash buffers.

🚫 Avoid

  • Do not use concentrators with < 30 kDa MWCO when using buffers from the scale-up kit, as they will likely retain and concentrate detergents. This could cause problems with solution phase separation, and impact the quality of recovered concentrated protein.