Technology Sharing Comprehensive Guide to Antibody Preservation

NOTE: Be sure to review the specific data on your antibodies for saving advice. We are not responsible for improperly stored antibodies. If stored and handled properly, the activity of most antibodies can be retained for months or even years.

save temperature

Freezing at -20°C or -80°C is the best storage condition for many antibodies. Split antibody can minimize the damage caused by freezing and thawing, and also avoid the introduction of contamination by removing antibody from the same tube multiple times. After repackaging, the antibody can only be frozen and thawed once. If there is any surplus, it should be kept at 4 ℃.

When you receive the customized antibody, centrifuge at 10,000 ×g for 20 seconds, settle the solution trapped in the tube cap threads, and then divide the antibody and transfer it to a low-protein binding microcentrifuge tube. The amount of antibody you can split depends on the amount you use in your experiment. When subpackaging antibody, each tube should be not less than 10 μL; The less the amount of subpackaging antibody, the more likely the concentration is to be affected by evaporation and adsorption on the surface of the storage sample tube.

In most cases, the antibody can be stored at 4°C for 1 to 2 weeks immediately after receipt. It is very important to follow the saving advice on the data sheet.

Special case

Do not cryopreserve enzyme-coupled antibodies, but store them at 4°C. Freezing and thawing will not only affect the binding ability of the antibody, but also reduce the enzyme activity.

Conjugate antibodies (whether conjugated with fluorochrome, enzyme, or biotin) should be stored in dark vials or wrapped in foil. Exposure to light can affect the activity of the conjugate. Fluorescent conjugates are particularly susceptible to photobleaching and should therefore be stored in the dark at all stages of the experiment.

IgG3 isotype antibodies are unique in that they readily form aggregates after thawing and should therefore always be stored at 4°C.

The ascitic fluid may contain protease, so it should be frozen as soon as possible after receipt.

Pollution

To prevent microbial contamination, sodium azide may be added to the antibody preparation at a final concentration of 0.02%(w/v). Antibodies typically shipped to the laboratory contain this protective agent in concentrations ranging from 0.02 to 0.05 percent. This is explained by the detailed data in the Save Buffer section.

The case of not using sodium azide

If living cells are to be stained or treated with antibodies, or if antibodies are to be used for in vivo studies, be sure to use preparations that do not contain sodium azide. This antibacterial agent has a toxic effect on most organisms: it blocks the cytochrome electron transport system.

Sodium azide interferes with all coupling reactions involving amine groups, so it must be removed before proceeding with the coupling reaction. After completion of the coupling reaction, the antibody can be preserved using sodium azide. Another antibacterial agent that can be used is 0.01% sodium thiomeralate (thimerosal), which does not contain primary amines.

The sodium azide in the antibody solution can be removed by dialysis or gel filtration. IgG has a molecular weight of 150,000 Da(IgM approximately 600,000 Da) and sodium azide has a molecular weight of 65 Da. A microdialysis device with a molecular weight cut-off of 14,000 Da allows the azide to diffuse out smoothly, while the antibody is retained.

The dialysis process was carried out in a beaker (placed on a magnetic stirrer, the temperature was maintained at 4°C) using at least 1 liter of pre-chilled PBS per ml of antibody, and the dialysis device was stirred for 6 hours. The PBS was changed twice with stirring for at least 6 hours after each change. If possible, all materials should be sterilized, and the resulting preparations should be handled under sterile conditions.

freeze-thaw damage

Repeated freezing and thawing may denature the antibody, causing the antibody to form aggregates, thereby reducing its binding ability.

For most antibodies, storage at -20°C is sufficient; storage at -80°C has no significant advantage. Frost-free refrigerators should be avoided, as the freeze-thaw cycles of such refrigerators (used to reduce frost formation) are precisely what needs to be avoided to preserve such antibodies. For the same reason, antibody vials should be placed in the area of the refrigerator with minimal temperature fluctuations, such as against the back of the refrigerator instead of on the refrigerator door frame.

Some researchers add the cryoprotectant glycerol (final concentration of 50%) to the antibody to prevent freeze-thaw damage; glycerol reduces the freezing point to below -20°C. Although this method can be used for a variety of antibodies, it is not recommended to store solutions containing glycerol at -80°C because this temperature is below the condensation point of glycerol. Please note that glycerol may be contaminated with bacteria. If glycerol or any cryoprotectant is added, care should be taken to ensure that a sterile preparation is obtained.

Protein concentration and stability

After diluting the antibody to the working concentration, it should be stored at 4°C for no more than one day. In general, proteins stored at high concentrations are less susceptible to degradation, with the ideal concentration being 1 mg/mL or higher. This is the rationale for adding a protein, such as BSA, to an antibody solution as a stabilizer. The added protein also minimizes losses due to binding of the antibody to the container walls. Do not add a protein stabilizer to the antibody to be conjugated, because the protein stabilizer will compete with the antibody and reduce the coupling efficiency.