Stabil-P.A.C. NV10 Removal

NVoy polymers interact with hydrophobic patches on a protein forming a polymer : protein complex in dynamic
equilibrium with the free polymer in solution. NV10 can be removed from a protein solution by binding the target
protein to ion-exchange or affinity media and washing out NV10 before elution of the target protein.

Ion exchange chromatography (IEx) is a technique whereby a charged protein can reversibly interact with an
oppositely charged matrix, and then be eluted by a change in buffer ionic strength or pH. Each protein is composed
of a unique combination of individual amino acids, and different proteins therefore often have differing net charges
at any given pH.

        • The pH at which a protein has no net charge is termed the iso-electric point (pI).
        • The pI of a protein should be determined experimentally, but if the amino acid sequence is known then the
            theoretical pI can be estimated at websites such as http://us.expasy.org/tools/ .
        • Under conditions where the pH is higher than the pI the protein will have a net negative charge, and bind to
            an anion exchange resin (positively charged).
        • Under conditions where the pH is lower than the pI the protein will have a net positive charge and bind to a
            cation exchange resin (negatively charged).

Affinity chromatography is a method of separating a mixture of proteins by utilizing specific interactions of the target
protein with a ligand which is immobilized on a support, eg IMAC, Protein A.

Troubleshooting
In most cases the target protein will bind as normal to the resin of choice. If the binding is weaker than expected
the interaction with the solid phase can be enhanced using a release agent to weaken the interaction between the
protein and NV10 polymer. Novexin supplies these release agents as part of Stabil-P.A.C.:

        • Addition of dimethylsulfoxide (DMSO) will facilitate a slow gentle release.
        • Addition of “NV10 removal solution” will facilitate instantaneous release.

PROTOCOL FOR RELEASE OF NV10
 

        1. Determine quantity of NV10 in solution.
        2. Up to 10 % of release agent (either DMSO, or 1X removal agent) may be added to a 1X solution of NV10
            (2.5 mg/ml).
        3. Incubate for 10 minutes room temperature.
        4. Perform NV10 removal using IEx or affinity chromatography.

Note! A protein which is intrinsically prone to aggregation or instability may precipitate at this point.

Troubleshooting
If a heavy protein precipitate forms at the release step:

       
        • Use DMSO as a release agent rather than “NV10 removal solution” to give a more gentle dissociation of
            components.
        • Add less release agent.
        • Add release agent in small aliquots over a longer period of time.
        • Perform release at 4 oC.

If NV10 is still present in the protein sample after release and removal:

        • Extend column chromatography wash step to 20 column volumes before protein elution.
        • Add 10 % DMSO to the IEx or affinity wash buffer before protein elution.