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FAQs

 
Custom antibody production
How do I store my antibody?
Storage Suggestion for Antibodies used for in vitro Research Applications

Properly stored, antibodies should remain stable for months or years. Freezers with automatic de-frosting are absolutely inappropriate for antibody storage.

If you are using catalogue antibodies, please refer to the provided data sheet.

Storage Temperatures

Our purified antibodies are delivered to you in PBS with 0,1% BSA as stabiliser and 0,01% Thimerosal as preservative. For these formulations we suppose that storage of small aliquots at –20°C is appropriate.

The aliquoting of antibodies has the following advantages:
1. Minimisation of the damage due to freezing and thawing
2. Exclusion of contamination due to multiple pipetting from a single vial
Aliquots should be thawed once, with any remaining antibody kept at 4°C.
When you receive an antibody you should carefully thaw it (in the fridge in a 4°C water bath – do not forget about it) and centrifuge it with 10.000 x g for 20 sec in order to pull down all the solution. Aliquot the antibody in low-protein-binding micro centrifuge tubes for storage. Please adapt the size of the aliquots to your typical experimental consumption. Aliquots should not be smaller than 10 µl to avoid damage of the antibody by evaporation and adhesion to the vessel wall.

Though it is considered in general as optional, we recommend the addition of 50% glycerol (v/v, final) to your antibodies for additional freezing and evaporation protection for storage at –20°C (glycerol is frozen at –80°C).

Please use just sterile chemicals and solutions for your antibodies to prevent down stream microbial growth.

Upon receipt, antibody storage for one or two weeks (time required for experimental establishment) at 4°C should be acceptable with subsequent aliquoted freezing for long-term storage.

Ascites and serum represent an exception here since the contained proteases require immediate storage at -20°C or -80°C.
Please be advised that sera produced at Eurogentec do not contain any preservative, 0,01% azide or thimerosal (depending on your research purpose) should be added to the aliquots for storage at –20°C after first thawing.

Egg yolks in contrast must never be frozen, since they denature into a form that does not allow isolation of IgY and affinity purification anymore. Egg Yolks from Eurogentec are shipped at 4°C with 0,01% azide added as preservative ready for short-term storage (over several weeks) at 4°C.

For long-term storage we recommend the purification of IgY from the egg yolks.

IgY isolates and affinity purified IgY antibodies should be treated like purified antibodies described in the beginning of this section.

Antibody formulations that require special attention:

  • enzyme-conjugated antibodies should not be frozen at all in order to retain the maximum enzymatic activity, storage at 4°C is therefore mandatory, please refer to the technical datasheet on the solvent formulation, and add if required 0,01% Thimerosal (Azide will inactivate HRP-conjugates)
  • all conjugated antibodies (enzymes, fluorophores, or biotin) should be stored in the dark (very well suited: the black foil bags of x-ray films or photographic paper, and the corresponding boxing) 
  • Fluorophore-conjugated antibodies can be stored aliquoted at –20°C with 50% glycerol (final v/v) – please make sure by the data sheet that they are formulated as well with BSA, physiologically concentrated buffer salts (e.g. PBS), and preservatives (azide or thimerosal), furthermore, please be sure to use a sterile glycerol formulation, since microbial growth might occur and destroy the antibody. Storage at –80°C of glycerol containing solutions is not advised since this is below the freezing point of glycerol
  • Minimisation of aggregate formation of some IgG isotypes (IgG3 for instance) requires storage at 4°C
Contamination prevention

Purified custom-made antibodies from Eurogentec already contain 0,01% thimerosal to avoid microbial growth, as mentioned on the data sheet. But please be advised that any serum, like Test Sera for animal selection, pre-immune sera (PPI), large bleed sera (GP), or final bleed sera (SAB) do not contain any preservative.

Please be advised of the following prior to use sodium azide for storage
  • azide is toxic to organisms and should be avoided in applications which are done with living cells, or tissues (the same is the case for thimerosal – please advise us before starting your immunisation programme of your research purpose in order to avoid any preservative)
  • if antibodies should be conjugated or spotted involving amino groups, azide will interfere negatively into the process, thimerosal is an acceptable alternative without these unwanted side effects
Preservatives, if already in an antibody formulation, can be removed in the following ways:
  • by dialysis or centrifugal filtration: using a device with a MWCO of 14 kDa, please refer to the manufacturer’s recommendations for most efficient use centrifugal filter devices or dialysis equipment (MW of sodium azide ~ 65 D, thimerosal ~ 404 D, IgG ~ 160 kDa, IgM ~ 600 kDa).
  • by gel filtration (which is faster than dialysis). Please refer to the manufacturer’s recommendations for most efficient use of suited filter cartridges 
  • please be advised that all materials used here should be sterile to ensure the subsequent stability of the antibody, and to avoid contamination by purification
Damage caused by freezing and thawing of antibodies

The antibody’s binding capacity can be reduced by aggregate formation due to denaturation following cycles of freezing and thawing.

Though a lot of researchers keep their antibodies at –80°C, there is no real explanation why –20°C might be inappropriate. In any case, antibodies should rather be stored protected from daily routine in the freezer, e.g. rather in the back than in the front, or in drawers than standing free accessible.

Working concentrations of antibodies should not be stored for more than one day at 4°C, since proteins are more susceptible to degradation if they are stored in lower concentrations. Ideal would be to formulate the antibody with additional protein, like BSA or milk powder to reach concentrations of total protein in the mg/ml range. BSA or milk powder in working solutions also minimizes antibody loss due to attachment to the vessel wall.

The addition of stabilizing proteins is not recommended for antibodies that are subject to conjugation or array spotting.

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