1	Determine the starting protein concentration (using eg. Expedeon’s BradfordUltra assay, BCA assay, absorbance at 280nm).
2	Typically a fivefold excess, by mass, of NV10 will protect the target protein.  For example, use 100 µg/ml NV10 for 20 µg/ml protein.
3	Each Stabil-P.A.C. tube contains 10mg NV10 as a lyophilised powder (40mg per tube in a Stabil-PAC MAXI).
4	Add the protein solution to NV10 in Stabil-P.A.C. tubes to get the desired concentration, or make up a stock solution (e.g. 5 mg/ml NV10) by adding buffer or distilled water to each Stabil-P.A.C. tube and then add this stock to the protein solution.
5	Continue with PD10 desalting of protein + NV10 solution as normal.
6	NV10 will co-elute with the protein in solution to give continuing protection downstream.
7	NV10 stock  solutions (up to 10 mg/ml) can be stored for up to 1 week at 4oC or for longer term at -20 oC.  More concentrated stock solutions should be used immediately.

Troubleshooting

A stock solution of 1 mg/ml BSA in 50 mM Tris, 0.15 M NaCl pH 8.0 (TS buffer) was prepared, along with a 1X solution of NV10 (2.5 mg/ml).  Samples were prepared in duplicate containing either 10 µg/ml of BSA in TS buffer alone or 10 µg/ml of BSA in TS buffer containing 100 µg/ml NV10.  2.5 ml of each sample was loaded onto a PD10 column according to the manufacturer’s  protocol, and eluted in 3.5 ml of TS buffer.  The total protein recovered was measured using Expedeon’s BradfordUltra solution.

Table 1:  BSA recovery after PD10 desalt column

Proteins are often lost due to non-specific binding, especially at low working concentrations, and even a “model” protein such as BSA can experience up to 15 % loss of protein on a PD10 desalting column.  Addition of 100 mg/ml NV10 minimises non-specific binding, and enables virtually full recovery.

Summary

NV10 can improve protein recovery from PD10 desalting columns.