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Detailed procedure for sodium azide removal from antibody solutions.
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 to be used for cell culture assays or conjugation, sodium azide removal from the antibody solution is recommended.
The following three procedures can be used to remove sodium azide.
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.
If possible, all materials should be sterilized and the resulting preparation handled aseptically. Cold conditions are recommended as the antibody is no longer protected by preservative.
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.
Kits are commercially available for purification of antibodies which can also be used to remove azide.
The following Abcam kits can be used to purify the antibody from a solution containing BSA, glycine, Tris or sodium azide. It can also be used to purify antibodies from crude samples such as ascites fluid or immune serum.
The method involves capture of the antibody on a Protein A resin and removal of unwanted substances by a simple wash procedure, which can be performed using a standard microfuge. The purified product is then eluted and neutralized.