Sodium Azide Removal Protocol

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

• Dialysis membrane/tubing of appropriate diameter and length to accommodate the sample volume (10-30 kDa MW cut off)
• Dialysis buffer: PBS
• Large beaker
• Magnetic stirrer at 4°C
• Stirring bar

Methods

1. Assemble the dialysis unit. Pre-condition the unit for a minimum of 1-2 min in the dialysis buffer to allow the membrane to hydrate.
2. Transfer the antibody solution into the dialysis unit.
3. Place the dialysis unit into a suitably sized beaker containing at 500mL-1L of dialysis buffer against which the antibody is to be dialyzed.
4. Place the beaker on a magnetic stirrer. Dialyze for a minimum of 1 h at 4°C.
5. Change the buffer 3-4 times and dialyze again for at least 2 h each time. Repeat until the desired number of buffer changes has been achieved.

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

Reagent and Material

• G25 spin column
• Equilibration buffer
• Centrifuge

Methods

Note: If commercially available purification columns are being used, please refer to the manufacturer's instructions for use.

1. Remove the cap from the spin column. Centrifuge at 1,000 g for 2 min to remove the storage solution.
2. Put the column in a collecting tube.
3. Fill the column with equilibration buffer. Centrifuge at 1,000 g for 2 min.
4. Repeat 3 times. Discard the collected flow-through.
5. Add 1-3 mL of antibody sample slowly to the middle of the packed bed and centrifuge at 1,000 g for 2 min.
6. Collect and recover the eluate (antibody) located in the collection tube.

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

BrdU Protocol

Primary Cultures for IHC-Viability Assays

Biotin Conjugation