Sodium azide removal

Last edited Thu 18 May 2023

Sodium azide is a preservative used for inhibiting the growth of contaminants, such as bacteria or fungi. However, its presence in antibody solutions can affect the use of the antibody in cell culture assays as it is toxic to cells. It can also interfere with antibody conjugation and inhibits the activity of the enzyme horseradish peroxidase.

Many Abcam antibody products contain sodium azide, and this information is provided on individual datasheets. If the antibody is used for cell culture assays or conjugation, sodium azide removal from the antibody solution is recommended.

Alternatively, if you're looking for a ready solution, check out our wide range of carrier-free recombinant antibodies, which are conjugation-ready and free from BSA, sodium azide, and glycerol.

The following three procedures can be used to remove sodium azide.

Assemble the dialysis unit, as recommended by the manufacturer. Pre-condition the unit for a minimum of 1–2 min in the dialysis buffer to allow the membrane to hydrate.

timelineNumber

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Stage 1 - Dialysis

A dialysis unit can be used to remove sodium azide from samples of 0.1 mL to 70 mL in volume. This is a semi-permeable membrane available in a wide range of size dimensions and pore sizes. Using a membrane with a pore size cut-off at 10-30 kDa will allow the azide to pass through the membrane but will retain the antibody and other proteins in the solution.

The molecular weight of IgG is 150 kDa (IgM is ~600 kDa). The molecular weight of sodium azide is 65 Da.

​A dialysis unit can be used to remove sodium azide from samples of 0.1 mL to 70 mL in volume. This is a semi-permeable membrane available in a wide range of size dimensions and pore sizes. Using a membrane with a pore size cut-off at 10-30 kDa will allow the azide to pass through the membrane but will retain the antibody and other proteins in the solution.

Assemble the dialysis unit, as recommended by the manufacturer. Pre-condition the unit for a minimum of 1–2 min in the dialysis buffer to allow the membrane to hydrate.

Transfer the antibody solution into the dialysis unit.

Place the dialysis unit into a suitably sized beaker containing at least 1 L of buffer against which the antibody is to be dialyzed. Place the beaker on a magnetic stirrer and dialyze for a minimum of 1 h at 4°C.

Change the buffer and dialyze again for at least 1 h. Repeat until the desired number of buffer changes has been achieved. Ensure that the buffer is changed at least 3 times.

Stage 2 - Desalting

This procedure is suitable for smaller volume of 1–3 mL. Desalting resins have size exclusion properties and consist of small particles with a range of pore sizes.

Size exclusion is a method used to separate molecules in solution by their molecules in solution by their molecular weight. Particles of varying molecular weight will elute through a size exclusion matrix at different rates. For example, large molecules cannot enter the pores of the matrix and therefore are eluted first, whereas smaller molecules will penetrate the pores within the beads and elute later.

A Sephadex G25 column system or equivalent will effectively remove sodium azide from an antibody sample. Pre-packed Sephadex spin columns are readily available and can be used for this procedure.

Materials required

• Sephadex G25 spin column
• Equilibration buffer
• Centrifuge

Remove the cap from the spin column and centrifuge at 1,000 x g for 2 min to remove the storage solution.

Put the column in a collecting tube.

Fill the column with equilibration buffer as advised by the manufacturer and centrifuge at 1,000 x g for 2 min.

Repeat 3 times and discard the collected flow-through.

Add 1–3 mL of antibody sample slowly to the middle of the packed bed and centrifuge at 1,000 x g for 2 min.

Collect and recover the eluate (antibody) located in the collection tube.