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NVoy Technology Improves Protein Recovery on PD10 Desalting Columns

 

Protein desalting is generally a high-yielding procedure, however protein losses do regularly occur when working
with sticky, aggregation-prone or dilute protein solutions. The addition of NV10 to such solutions can considerably
reduce losses onto column media resulting in improved yields.


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 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. 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 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 that have no history of            

        aggregation.

 


EXAMPLE : Use of NV10 With PD10 Columns

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). This was prepared by adding 500 μl of TS buffer to one Stabil-P.A.C. tube. 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 Novexin’s Bradford ULTRA solution.


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 μg/ml NV10 minimises non-specific
binding, and enables virtually full recovery.

 

Summary
NV10 can improve protein recovery from PD10 desalting columns.
 

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