Stabil-P.A.C. NV10 Improves Protein Recovery after Dialysis

Protein dialysis is a useful technique to enable a gentle change in protein buffer composition, the adjustment of salt
and additive concentrations or the removal of low molecular weight impurities. Potential disadvantages of the
technique include the loss of protein due to non-specific binding onto the dialysis membrane, and loss due to
protein aggregation and instability during the intermediate stages of buffer exchange. The addition of NV10 to
proteins before dialysis can minimise protein losses due to these factors and help to maximise protein recovery.
NV10 is a large polymer which associates with the target protein and protects it from aggregation. Although NV10
is in dynamic equilibrium with the protein its large size means that it can generally be retained within the dialysis
membrane and is therefore not required in the dialysis buffer.

PROTOCOL
Aggregation and stability are very protein specific, but a general protocol is given below.

    1. Determine the starting protein concentration (using eg. Bradford 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 1.25 mg NV10 as a lyophilised powder.
    4. Add the protein solution to NV10 in Stabil-P.A.C. tubes to get the desired concentration, or make up a
        2.5 mg/ml stock of NV10 (1X stock) by adding 500 μL of buffer or distilled water to each Stabil-P.A.C.
        tube and add this stock to the protein solution.
    5. Dialyse this protein / NV10 solution.
    6. NV10 associates with the protein in solution and is retained by the membrane during dialysis to give
        continuing protection further downstream.
    7. NV10 1X stock solution can be stored for 1 week at 4 oC or for longer term at -20 oC.

Troubleshooting
    • If the protein shows signs of aggregation or heavy losses the relative NV10 concentration can be
        increased, ie increase NV10 concentration and / or reduce protein concentration.
    • Alternatively, a lower NV10 to protein ratio can be used with proteins that have no history of aggregation.
    • Protein solutions dialysed using membranes with pore sizes of greater than 10,000 kDa may gradually
        lose NV10 into the dialysis buffer.

EXAMPLE : Use of NV10 in Dialysis
A stock solution of 1 mg/ml β-lactoglobulin in 50 mM Tris, 0.15 M NaCl pH 8.0 (TS buffer) was prepared, along with
a 2.5 mg/ml solution of NV10. This was prepared by adding 500 μl of TS buffer to one Stabil-P.A.C. NV10 tube.
Samples were prepared in duplicate containing 10 μg/ml of β-lactoglobulin either in TS buffer alone, or in TS buffer
containing either 10 μg/ml or 100 μg/ml NV10. 2 ml of each sample was loaded into a Pierce Slide-A-Lyser dialysis
cassette (10,000 mwco) according to the manufacturer’s protocol, and dialysed overnight at 20 oC against TS
buffer. The volume of the protein recovered was measured and the protein concentration was estimated using
Novexin’s Bradford ULTRA solution.

The protein losses associated with dialysis decrease in the presence of increasing NV10 concentrations. Full
recovery of β-lactoglobulin was achieved after dialysis in the presence of 100 μg/ml NV10.

Summary
NV10 can protect proteins from aggregation and loss to membranes during dialysis.