Abcam Antibody Preservation Guide - Huaqiang Electronic Network

This guide is based on the abcam antibody preservation method, and can also be applied to antibody products of most other companies! Please check the antibody data sheet from time to time for specific preservation recommendations. Storage Temperatures and Conditions For many antibodies, fractionation into small aliquots and cryopreservation at -20 ° C or -80 ° C is the best storage conditions. Dispensing into small aliquots minimizes damage caused by freeze-thaw and contamination introduced by multiple draws from a single reagent bottle. Small aliquots need only freeze and thaw once, and if left, the residue can be stored at 4 °C. Upon receipt of the antibody, centrifugation was carried out at 10,000 xg for 20 seconds to deposit a solution trapped in the reagent bottle thread and transferred to a low protein binding microcentrifuge tube in small aliquots. The amount of small aliquots depends on the amount that the experimenter typically uses in the experiment. The small aliquot should be no less than 10 μl; the smaller the aliquot, the greater the concentration of the stock solution due to evaporation of the antibody and adsorption to the surface of the storage bottle. In most cases, it is acceptable to store the antibody at 4 ° C for one to two weeks and then freeze for long-term storage, but perhaps the ascites solution, which may contain proteases, should be frozen as soon as possible. Again, it is important to follow the recommendations on the data sheet. Other special conditions: Enzyme-conjugated antibodies should not be stored frozen, but should be stored at 4 °C. Freezing and thawing not only affects antibody binding capacity, but also reduces enzyme activity. Whether coupled to a fluorescent dye, enzyme or biotin, the conjugated antibody should be stored in a dark reagent bottle or wrapped in a metal foil. Exposure to light will compromise the activity of the conjugate. In particular, fluorescent conjugates are susceptible to photobleaching and should be protected from light at all stages of the experiment. IgG3 isotype antibodies have a unique tendency to form aggregates upon thawing and should always be stored at 4 °C. Prevention of contamination with sodium azide To prevent microbial contamination, sodium azide can be added to the antibody preparation to a final concentration of 0.02% (w/v). Many abcam antibodies already contain such preservatives in concentrations ranging from 0.02 to 0.05%.
In the absence of sodium azide: If staining or treating live cells with antibodies, or if in vivo studies with antibodies, be sure to use preparations that do not contain sodium azide. The antibacterial agent is also toxic to most other organisms: it blocks the cytochrome electron transport system. Any coupling of sodium azide that interferes with the participation of the amino group should be removed prior to coupling. After coupling, the antibody can be stored in sodium azide. In addition, 0.01% thiomersal (thiomersal) contains no primary amine and is an acceptable substitute. Sodium azide can be removed from the antibody solution by dialysis or gel filtration. The molecular weight of IgG is 150,000 Daltons (IgM is about 600,000); the molecular weight of sodium azide is 65 Daltons. A microdialysis device with a molecular weight cutoff of 14,000 daltons retains the antibody while allowing the azide to diffuse outward. In a beaker kept on a magnetic stirrer at 4 ° C, at least one liter of cold PBS was used per ml of antibody and the dialysis apparatus was stirred for 6 hours. Replace the PBS twice and stir for at least 6 hours each time. All materials should be sterilized if possible and the resulting preparation should be handled aseptically. Freeze/Thaw Damage Repeated freeze/thaw cycles can denature antibodies, causing them to form aggregates that reduce antibody binding capacity. Preservation at -20 °C is appropriate for most antibodies; there is no significant advantage in storage at -80 °C. The refrigerator cannot be frost-free. These cycles between freezing and thawing (to reduce frosting) should be avoided. For the same reason, the antibody reagent bottle should be placed in the area of ​​the refrigerator with minimal temperature fluctuations, such as toward the rear of the refrigerator rather than on the gantry. Some researchers added cryoprotectant glycerin to a final concentration of 50% to prevent freezing/thawing damage; glycerol reduced the freezing point below -20 °C. Although this is acceptable for many antibodies, only a small percentage of the antibodies we provide test stability under this storage condition, and our assurances are only used for antibodies that are preserved in the manner recommended on the data sheet. It is not recommended to store the solution containing glycerol at -80 ° C because this temperature is lower than the freezing point of glycerol. Please note that glycerin can be contaminated by bacteria. If glycerin or any cryoprotectant is added, care should be taken to obtain a sterile preparation. Antibodies should be avoided at 4 ° C for more than one day after dilution to working concentration. Proteins are generally not easily degraded when stored at high concentrations, ideally at a concentration of 1 mg/ml or higher. This is the theoretical basis for the addition of proteins such as BSA as a stabilizer in antibody solutions. The added protein also helps to reduce the loss of antibodies due to binding to the walls of the container. For antibodies intended for conjugation, stable proteins should not be added since stable proteins compete with antibodies and reduce the efficiency of conjugation.