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Guidelines for the storage of different types of antibodies, avoiding contamination or damage.
Updated September 22, 2022
With proper storage and handling, most antibodies should retain activity for months, if not years. Here we provide general guidelines on antibody storage and handling. Please always refer to the manufacturer's datasheet for specific storage recommendations.
Upon receiving the antibody, you will need to centrifuge it at 10,000 x g for 20 seconds to pull down the solution trapped in the vial threads and then aliquot it into low-protein-binding microcentrifuge tubes. Aliquotting minimizes damage due to repeated freeze/thaw cycles that can denature an antibody, causing it to form aggregates that reduce its binding capacity. Aliquotting also helps minimize contamination introduced by pipetting from a single vial multiple times.
Aliquots should be frozen and thawed once, with any remainder kept at 4°C. It's generally recommended to store antibodies at -20°C as there's no significant advantage to storing them at -80°C. However, you should always check the antibody datasheet for specific temperature recommendations, as these might differ for certain antibody formats. For example, our BSA- and azide-free antibodies should be stored at 4°C and should not be frozen.
The size of the aliquots will depend on how much you typically use in an experiment. Aliquots should be no smaller than 10 μL. The smaller the aliquot, the more the stock concentration is affected by evaporation and adsorption of the antibody onto the surface of the storage vial.
In most cases, storage at 4°C upon receiving the antibody is acceptable for one to two weeks. It is essential to follow the recommendations on the datasheet.
Make sure not to use a frost-free freezer: it's unlikely your lab would, but the cycling between freezing and thawing should be avoided. For the same reason, antibody vials should be placed in the freezer area with 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 because glycerol lowers the freezing point to below -20°C. While this may be acceptable for many antibodies, you should check the datasheet to see if the manufacturer tested antibody stability in this storage condition.
Storing solutions with glycerol at -80°C is not advised since this is below the freezing point of glycerol. Also, glycerol or any other cryoprotectant can be contaminated with bacteria, so you must obtain a sterile preparation.
Conjugated antibodies often require additional storage and handling precautions since they're more complicated than non-conjugated antibodies. For example, conjugated antibodies – whether conjugated to fluorochromes, enzymes, or biotin – should be stored in dark vials or wrapped in foil because exposure to light will compromise conjugates' activity. Fluorescent conjugates, in particular, are susceptible to photo-bleaching and should be protected from light during all phases of an experiment.
The table below provides detailed guidelines for proper conjugated antibody storage and handling.
Fluorescent labels, eg, Alexa Fluor®, Dylight®, FITC, PE
Enzyme-conjugated, eg, HRP
Aliquot upon delivery
Aliquot after gently mixing with a pipette. Repeat 3-4 times. Do not mix by inversion, as this may cause the solution to foam
Do not add sodium azide to HRP-conjugated antibodies since this preservative inhibits HRP activity
Follow the manufacturer's datasheet recommendations
Store at +4°C short term (1-2 weeks)
*Freezing and thawing enzyme-conjugated antibodies will reduce enzymatic activity and affect the antibody binding capacity. Therefore, enzyme-conjugated antibodies should not be frozen at all and should instead be kept at 4°C unless an antibody contains a cryoprotectant, and its stability has been validated for long-term storage at -20°C.
Avoiding contamination with sodium azide
To prevent microbial contamination, you can add sodium azide to an antibody solution to a final concentration of 0.02% (w/v). If an antibody already contains this preservative, this will be indicated on the datasheet in the storage buffer section.
When not to use sodium azide
Sodium azide should be avoided when staining or treating live cells with antibodies or conducting in vivo studies. This antimicrobial agent is toxic to most other organisms as it blocks the cytochrome electron transport system.
Sodium azide will interfere with any conjugation involving an amine group and should be removed before proceeding with the conjugation. After conjugation, you can store antibodies in sodium azide, except for HRP-conjugated antibodies, since sodium azide inhibits HRP. An acceptable alternative to sodium azide is 0.01% thimerosal (merthiolate), which does not have a primary amine. Also, sodium azide can be removed from antibody solutions by dialysis, ultrafiltration, or gel filtration.
Proteins are generally less susceptible to degradation when stored at higher concentrations, ideally ³1 mg/mL. Therefore, you should avoid diluting antibodies to working concentration and keeping them at 4°C for more than a day.
If the antibody concentration is low, stabilizer proteins such as BSA can be added to the antibody solution. The stabilizer protein also serves to minimize the antibody loss due to binding to the vessel wall. However, you shouldn't add stabilizing protein to the antibodies you intend to conjugate because it will compete with the antibody and reduce the conjugation efficiency.
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.
DyLight® is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries.
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