实验概要
Sodium azide is a preservative used for inhibiting the growth of contaminants such as bacterial or fungus in antibody solutions. 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 affect antibody conjugation and also inhibits the detection 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.
主要试剂
1. Dialysis Buffer e.g. PBS, TBS or HEPES (pH range 6 - 8).
主要设备
1. Large beaker .
2. Magnetic stir plate and stirrer kept at 4°C.
3. Dialysis membrane/ tubing (12,000 to 14,000 Dalton molecular weight cut-off).
实验步骤
Three main procedures can be used to remove azide:
1. Dialysis
This procedure is used to remove azide from the large volume (0.1ml to 30 ml) of samples. A micro-dialysis unit can be used for 0.1 ml to 1.5 ml antibody volume. This is a semi-permeable membrane available in a wide range of size dimensions and pore sizes. Using a membrane with pore size cut-off at 12,000 to 14,000 daltons will allow the azide to pass through the membrane by retaining antibody and other proteins in the solutions. (The molecular weight of IgG is 150,000 Daltons (IgM is ~ 600,000); the molecular weight of sodium azide is 65 Daltons.
Procedure
1) Put together the dialysis unit, as recommended by the manufacturer. Check this for leaks by adding appropriate buffer before starting.
2) Transfer the antibody solution to a pre-wetted dialysis unit of the appropriate diameter and length to accommodate the volume.
3) Place the dialysis unit into a suitably sized beaker containing the buffer (PBS) against which the antibody is to be dialyzed. Keep the PBS containing beaker at 4°C.
4) Dialyze by using at least a liter of cold PBS per ml of antibody and stir (using a magnetic stirrer) the dialysis unit for 6 hrs per exchange. Change the buffer at least twice at convenient intervals. If possible, all materials should be sterilized and the resulting preparation should be handled aseptically. Cold temperature is required because the antibody is no longer protected by preservative.
2. Centrifugation
This procedure is suitable for smaller volumes 1 – 3 ml based on spin columns. The principle of this procedure is based on the fact that molecules larger than the largest pores in a Sephadex matrix cannot enter in pores of the matrix and therefore eluted first. Molecules smaller than the largest pores will penetrate the pores within the Sephadex beads such that they have to go through larger accessible column volume than the large molecules and therefore they elute after the large molecules just before one total column volume of buffer has passed through the column.
Sephadex G25 column system will effectively remove the azide as this will be eluted from the Sephadex in solution later than the antibody does. These pre-packed spin columns are easily commercially available and can be used for this purpose.
Procedure
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 and pour off the storage solution.
2) Put the column in a collecting tube.
3) Fill the column with equilibration buffer or the buffer as advised by manufacturer and spin down at 1000 g for 2 minutes.
4) Repeat the step 3 times and discard the flow-through.
5) Add 2-3 ml of sample antibody slowly in the middle of the packed bed.
6) Elute the sample by centrifugation 1000 x g for 2 minutes.
7) Collect the eluate.
3. Antibody purification kits
Kits are commercially available for purification of antibodies which can also remove the azide. The following Abcam kits can be used to purify the antibody from the solution containing BSA, glycine, tris or sodium azide. These can also be used to purify antibodies from crude samples such as ascites fluid or immune serum. The method involves capturing of the antibody on protein A resin and then removal of unwanted substances by a simple wash procedure, which is carried out in a standard microfuge. The purified product is then eluted and neutralized.
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