Sodium Azide Removal Protocol

Licia Miller   Product Manager


Sodium azide is a commonly used preservative to prevent bacterial and fungal contamination in antibody solutions. However, sodium azide is cytotoxic, interferes with antibody coupling reactions, and inhibits the activity of enzymes such as horseradish peroxidase (HRP). Therefore, in some experiments (such as cell culture, antibody coupling, etc.), sodium azide needs to be removed from the antibody solution.

 

Many of Aladdin's antibody products contain sodium azide, the information of which is provided in the respective product datasheets. If the antibody is used for cell culture detection or conjugation, it is recommended to remove sodium azide from the antibody solution. Alternatively, you can also choose our carrier-free recombinant antibodies, which are free of BSA, sodium azide and glycerol and can be used directly.

 

This article will introduce three commonly used methods for removing sodium azide.

 

 

Method 1    Dialysis

 

Dialysis is a method that uses a semipermeable membrane to separate small molecules from large molecules. The molecular weight of sodium azide is 65 Da, while the molecular weight of antibodies (such as IgG) is about 150 kDa, so sodium azide can be removed by dialysis.

 

Dialysis devices are generally available in a variety of sizes and pore sizes. Here, we use a membrane with a pore cutoff of 10-30 kDa, depending on the size of the molecule to be separated, allowing the azide to pass through the membrane while retaining the antibody and other proteins in the solution.

 

1. Assemble the dialysis apparatus according to the manufacturer's recommendations and pre-wet the dialysis membrane with dialysis buffer for 1-2 min.

 

2. Transfer the antibody solution from which sodium azide needs to be removed to a dialysis device.

 

3. Place the dialysis apparatus in a large beaker and add at least 1 L of buffer to the beaker.

 

4. Place the beaker on a magnetic stirrer and dialyze at 4°C for at least 1 hour.

 

5. Replace the buffer in the beaker and dialyze again for at least 1 hour. Repeat at least 3 times.

 

Method 2    Desalting

 

The core principle of desalting is size exclusion chromatography (SEC), also known as gel filtration chromatography. This method uses the pore structure of gel particles to separate different substances according to molecular size.

 

Size Exclusion Chromatography (SEC) uses porous gel particles, such as Sephadex G25. These particles have a fixed pore size and are typically used to separate molecules with molecular weights ranging from a few hundred to a few thousand Daltons.

 

Macromolecules such as antibodies cannot enter the pores of gel particles due to their large molecular weight, so when passing through the gel column, they mainly move in the gaps between gel particles. Due to the shorter path, macromolecules will pass through the column faster and be eluted first.

 

Small molecules (such as sodium azide): Due to their small molecular weight, they can enter the pores of the gel particles. Therefore, when passing through the gel column, they need to go through a longer path, the elution speed is slower, and they are finally eluted.

 

The pore size of Sephadex G25 is just right for separating small molecules (such as sodium azide, molecular weight 65 Da) and large molecules (such as antibodies, molecular weight about 150 kDa) , and can effectively remove sodium azide from antibody samples.

 

If using a commercial purification column, please refer to the manufacturer's instructions . In other cases, refer to the following steps.

 

1. Open the cap of the spin column and centrifuge at 1,000 × g for 2 minutes to remove the storage solution.

 

2. Transfer the column to a collection tube.

 

3. Fill the column with equilibration buffer as recommended by the manufacturer and centrifuge at 1,000 × g for 2 minutes.

 

4. Repeat 3 times and discard the collected effluent.

 

5. Slowly add 1-3 mL of antibody sample to the middle of the purification column and centrifuge at 1,000 × g for 2 minutes.

 

6. Collect the eluate (antibody).

 

Method 3   Antibody Purification Kit

 

Directly purchase a commercial antibody purification kit and strictly follow the kit instructions.

 

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/