My affinity purified polyclonal antibody from rabbit serum is actually concentrated at 80 mg/mL in PBS with 0.05% Proclin C (as preservative). I would like to store it at 4°C, but I can not add any stabilizer agents, such as BSA or gelatin. Eventually, I would only use glycerol to render more stable the IgG. Any suggestions would be greatly appreciated.
Dear Sergio,
an important question! Thanks for asking.
Aliquot your antibodies and store them in different fridges/freezers and locations.
Snap-freezing them in liquid nitrogen and storing them at -80degC or lower might also an option.
5..20mg/ml should be fine. Check pH, it should be around neutral. If possible, use sodium azide instead of proclin (or just add NaN3 to 0.1% m/v final. To me, dilute proclin seems to degrade or at least loose its efficiency after some time). I have stored large quantities (100ml, 20mg/ml, in PBS/N3) of chicken IgY in the fridge for 1-2 years without apparent problems. Slight aggregations usually will dissolve upon dilution to concentrations below 5mg/ml. Consider titering your AB, to have reference data for quality control.
Untested by myself, but others rely on it: Could those in the know please comment:
- 50% glycerol and fridge are said not to match. Might kill your AB in short time
- Precipitate by (almost) saturating with ammonium sulfate, then store as is in the fridge
Is anyone aware of a paper/study on antibody storage?
I usually store my antibody stock in 50 % glycerol at -20. It can be very concentrated. The vial in use also can be kept at -20 in 50 % glycerol, I would just dilute it to a concentration of IgG, which is most convenient for your needs.
Agree with Fernando. 80 mg is far to concentrated, danger of aggregation.
My advice: store aliquots. Make dilutions starting at 5 mg/mL and do not go above 15 mg/mL. I believe 1 mg/mL is too little protein in solution and you might lose a lot of antibody in the walls of your tube. 80 mg/mL is too much and you run the risk of aggregation and having to denature your antibody before any use which is a real ordeal.
Dear Sergio,
an important question! Thanks for asking.
Aliquot your antibodies and store them in different fridges/freezers and locations.
Snap-freezing them in liquid nitrogen and storing them at -80degC or lower might also an option.
5..20mg/ml should be fine. Check pH, it should be around neutral. If possible, use sodium azide instead of proclin (or just add NaN3 to 0.1% m/v final. To me, dilute proclin seems to degrade or at least loose its efficiency after some time). I have stored large quantities (100ml, 20mg/ml, in PBS/N3) of chicken IgY in the fridge for 1-2 years without apparent problems. Slight aggregations usually will dissolve upon dilution to concentrations below 5mg/ml. Consider titering your AB, to have reference data for quality control.
Untested by myself, but others rely on it: Could those in the know please comment:
- 50% glycerol and fridge are said not to match. Might kill your AB in short time
- Precipitate by (almost) saturating with ammonium sulfate, then store as is in the fridge
Is anyone aware of a paper/study on antibody storage?
Than less is the concentration than shorter would be the storage time. It is a general rule that works in many cases including the antibodies.
HI,I usually store my antibodies (stock) in 50 % glycerol at -80C for long term and in different aliquots. It can be very concentrated at this stage. However, if it is an antibody that will be used quicly -20C is fine. We tend to use 1 aliquot at a time and dilute the antibodies to the appropriate IgG concentration depending on the application.
I agree with most of the answers but it is important to take into account that there could be a great deal of variability depending on the antibodies (after all their variable regions are unique) thus some are more resilent than others. I have had antibodies that were fine after a long time at 4ºC and some that perished easily. In any case 50% glycerol at -20 or -80ºC for some antibodies and/or longer periods should be fine. A good thing about it too is that you do not need to aliquot as it will bot freeze due to the high concentration of glycerol, thus you can always remove some for your working stock. The only thing to take into account is to have it concentrated enough so you can dilute it afterward to obtain a glycerol concentration that does not hamper your specific application.
@Curro - Aliquots
Consider always to make aliquots. Repeated opening/closing of vials is a risk for contamination (condensing water, dust particles, impurities, microbes). Other risks are loss, misplacing, labels dropping off/becoming unreadable, fridge/freezer failure.
Even when your antibody is sort of homebrew and you maybe have gram quantitites, they usually are quite precious in terms of work, time and money.
Thre is e eutectipc point in protein solution which is normally between -15 und -25 °C. If you are stay below this -25 °C (you can -30 ° or lower) use you even can keep protein solutions of 0.5 mg/mL stable. Be careful, if you are storing monoclonal antibodies, they are sometimes sensible. The are drifting to self-aggregations. On the other hand, you can keep them away from this self-aggregation only at low concentrations. Thats the reason why it's helpfult to store low antibody concentrations (below 0.5 mg/mL) under protein protection (e.i. 0.5 ... 1% BSA).
Why storing below the eutectic point: at this temperature recrystallization processes are ongoing whenever you are passing this temperature. At the surface of the water crystals starting denaturation processes due to the high salt concentrations ...
PS: Storage in low volume aliquots is frequently better than storing larger volumes in gylcerol
Thank you very much to everybody for helping me. Based on your suggestions, I decided to flash freeze small aliquots of my polyclonal antibodies diluted 5-10 mg/mL in liquid nitrogen and to store them at -80°C.
Well done Sergio ! Glad to be helpful ! Wish you the best. Share the pics of your blots when you get awesome results !
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