Biotinylating AviTag™ labelled proteins produced with eProtein Discovery™ using BirA
This protocol describes the process of biotinylating AviTag™-labelled proteins produced with eProtein Discovery™ using biotin ligase (BirA). The AviTag™ sequence must be introduced into the gene-of-interest, located between the 3C and TEV adaptor sequences.
Reagents and Materials required
| Item | Supplier | Product Code |
|---|---|---|
| Elution Buffer (provided in Scale up kit) | Nuclera | NC3011 |
| BirA Biotin Ligase (recombinant, E. coli, ≥65%) | Merck | SRP0417-100UG |
| ATPSolution (100 mM) | ThermoScientific | R0441 |
| MgCl2 (0.2 M prepared in MilliQ Water) | - | - |
| Zeba™SpinDesalting Columns, 40K MWCO | ThermoScientific | A57762 |
Method
Aminimumproteinconcentration of 4 μM in elution buffer is recommended. If the protein concentration is lower, concentrate it using a spin concentrator with a molecular weight cutoff appropriate for the protein's size (not below 30 kDa).
Prepare BirA
- Prepare a 10 μMstock solution of BirA in elution buffer. Note: This can be stored aliquoted at-80 oC.
- Dilute BirA to a working concentration that is 2.5X less than the concentration of the AviTag™-labelled protein.
Biotinylating AviTagTM labelled proteins
- Set up the biotinylation reaction in a 1.5 mL microcentrifuge tube according to Table 1.
-
- Incubate the reaction for 2 hours at 30°C.
| Item | Volume |
|---|---|
| Avi-Tag™ Protein (minimum concentration 4 μM) | 500 µL |
| Diluted BirA (Concentration 2.5x less than protein) | 5 μL |
| MgCl2 (0.2 M) | 15 μL |
| ATP (100 mM) | 15 µL |
| Elution Buffer | 65 µL |
Removing excess biotin
Excess biotin is removed via buffer exchange using Zeba™ Spin Desalting Columns, 40K MWCO, following the manufacturer’s instructions. For sample volumes between 300–800 μL, use product A57762 and the protocol below. For alternative sample volumes, choose an appropriate Zeba™ Spin Desalting Column and follow the manufacturer’s guidelines.
- Remove the column’s bottom closure and loosen the cap (do not remove it completely).
- Place the column in a collection tube and centrifuge at 700 x g for 2 minutes to remove the storage solution.
- Discard the flowthrough and reposition the column in the collection tube.
- Add 1 mL of the buffer of choice to the top of the resin and centrifuge tube at 700 x g for 2 minutes. Discard the flowthrough.
- Repeat step 4 once.
- Add 1 mL of the buffer of choice to the top of the resin and centrifuge tube at 700 x g for 3 minutes. Discard the flowthrough. Note: Ensure the resin appears white and free of liquid after each spin. If liquid is present, verify the centrifugation speed and time.
- Transfer the column to a new collection tube.
- Apply the biotinylated sample on top of the resin (optional) For low sample volumes or with low concentration apply a 40 μL stacker to the top of the resin bed after the sample has fully absorbed to ensure maximal protein recovery.
- Sample recovery: Centrifuge at 700 x g for 3 minutes, collect the flowthrough containing the biotinylated sample, and discard the spin column. Additional Notes: Other spin columns have been tested and resulted in excess loss of protein. If preparing for Biacore SPR analysis, we recommend using Cytiva 1X HBS-P+ buffer.
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