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NV10 Improves Recovery after Protein Concentration
Protein aggregation is strongly dependent on protein concentration, and so the preparation of a concentrated
protein solution can be problematic with associated protein aggregation and membrane losses. The build-up of
protein aggregates on the surface of the membrane filter during filtration and the membrane polarization layer can
increase resistance to filtrate flow and decrease filtration efficiency. Novexin’s NV10 polymer protects the target
protein and stabilises the protein at increasing concentrations helping to keep it monomeric and soluble, as well as
minimising protein losses to hydrophobic surfaces and speeding up filtration.


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. Concentrate the protein / NV10 solution to the desired protein concentration.
    6. NV10 will co-concentrate with the protein in solution to give continuing protection as the protein
        concentration increases.
    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 NV10 to protein concentration ratio can be
        increased, ie increase NV10 concentration and / or reduce protein concentration.
    • Alternatively, a lower NV10 to protein ratio can be used with proteins which have no history of aggregation.


EXAMPLE: Use of NV10 in Protein Concentration
A stock solution of 1 mg/ml BSA in PBS was prepared, along with a 2.5 mg/ml solution of NV10. This was prepared
by adding 500 μl of PBS to one Stabil-P.A.C. tube. These stocks were used to prepare duplicate samples
containing 10 μg/ml BSA in PBS and supplemented with increasing concentrations of NV10. 1 ml aliquots of each
sample were placed into Vivaspin 2 spin concentrators (5,000 mwco, Hydrosart low protein-binding membrane) and
concentrated tenfold according to the manufacturer’s instructions. The concentrate was collected using reverse
spin, and the recovered protein content was measured using Novexin’s Bradford ULTRA reagent.

Increasing NV10 concentration during spin concentration of
BSA has a marked effect on recovery, with 100 μg/ml NV10
doubling the recovered yield of BSA.

Summary
NV10 can increase protein recovery after spin concentration.

 

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