Guidelines for the storage of different types of antibody

Guidelines for the storage of different types of antibody

Storage temperatures and conditions

For many of our antibodies, freezing at -20 C or -80 C in small aliquots is the optimal storage condition. Aliquotting minimizes damage due to freezing and thawing, as well as contamination introduced by pipetting from a single vial multiple times. Aliquots should be no smaller than 10 µl.

Upon receiving the antibody, centrifuge at 5,000 x g for 30 seconds to pull down the solution, and transfer aliquots into low-protein-binding microcentrifuge tubes. Antibodies should be frozen as soon as possible, storage at 4 C upon receipt of the antibody is acceptable for one to two weeks, followed by freezing for long-term storage.

To prevent microbial contamination, sodium azide can be added to an antibody preparation to a final concentration of 0.02% (w/v). If using antibodies for in vivo studies, please be sure to use preparations that do not contain sodium azide. This antimicrobial agent blocks the cytochrome electron transport system. Sodium azide will interfere with any conjugation that involves an amine group and should be removed before proceeding with the conjugation. After conjugation, antibodies can be stored in sodium azide but 0.01%  thimerosal (Merthiolate), which does not have a primary amine, is an acceptable alternative.

Sodium azide can be removed from antibody solutions by dialysis or gel filtration. The molecular weight of IgG is 150,000 daltons (IgM is ~ 600,000); the molecular weight of sodium azide is 65 daltons. A micro-dialysis unit with a cut off at 14,000 daltons will retain the antibody as the azide diffuses out. In a beaker on a magnetic stirrer kept at 4 C, use at least a liter of cold PBS per ml of antibody and stir the dialysis unit for 6 hrs. Change the PBS twice, stirring at least 6 hrs for each change. If possible, all materials should be sterilized and the resulting preparation should be handled aseptically.

Freeze/thaw damage

Repeated freeze/thaw cycles can denature an antibody, causing it to form aggregates that reduce the antibody’s binding capacity.

Storing at -20 C should be adequate for most antibodies; there is no appreciable advantage to storing at -80 C. The freezer must not be of the frost-free variety. These cycle between freezing and thawing (to reduce frost-build-up), which is exactly what should be avoided. For the same reason, antibody vials should be placed in an area of the freezer that has minimal temperature fluctuations, for instance towards the back rather than on a door shelf.

Some researchers add the cryoprotectant glycerol to a final concentration of 50% to prevent freeze/thaw damage; glycerol will lower the freezing point to below -20 C. While this may be acceptable for many antibodies, only a small percentage of the antibodies we offer have been tested for stability in this storage condition and our guarantee only applies to antibodies stored as recommended on the datasheet. Storing solutions containing glycerol at -80 C is not advised since this is below the freezing point of glycerol. Please be aware that glycerol can be contaminated with bacteria. If adding glycerol or any cryoprotectant, care should be taken to obtain a sterile preparation.

Diluting antibodies to working concentration and storing at 4 C for more than a day should be avoided. Proteins, in general, are less susceptible to degradation when stored at higher concentrations, ideally 1 mg/ml or higher. This is the rationale for including proteins such as BSA in the antibody solution as stabilizers. The added protein also serves to minimize loss of antibody due to binding to the vessel wall. For antibodies that one intends to conjugate, stabilizing proteins should not be added since they will compete with the antibody and reduce the efficiency of the conjugation.