Sodium azide is a preservative normally used for inhibiting the growth of contaminants such as fungi and bacteria in antibody solutions. However, sodium azide can interfere with antibody conjugation and inhibits the activity of the enzyme horseradish peroxidase (HRP). Its presence in antibody solutions can also affect the use of the antibody in cell culture assays because it is toxic to cells. Many commercial 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 suggested. The following approaches can be used to remove sodium azide, dialysis, desalting and antibody purification kits. It is worth noting that most methods will result in some loss of protein during transfer by adherence to surfaces.
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. The molecular weight (MW) of IgG and IgM is 150 kDa and 600 kDa respectively. The MW of sodium azide is 65 Da. 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.
Reagent and Material
Methods
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 MW. Particles of varying MV will elute through a size exclusion matrix at different rates. 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 method.
Reagent and Material
Methods
Note: If commercially available purification columns are being used, please refer to the manufacturer's instructions for use.
Antibody Purification Kit
Kits which are commercially available for purification of antibodies can also be used to remove azide. The method involves capture of the antibody on a Protein A resin and removal of unwanted substances by a simple wash step, which can be performed using a standard microfuge. The purified product is then eluted and neutralized.
Other Protocols
Antibody Staining of Whole Mount Drosophila Embryos
