Now that you have your antibodies stock organized it’s about time to use them!
Most laboratories utilize antibodies one way or another. The ability to generate antibodies to various molecules combined with their inherent high specificity and stability turned antibodies into the #1 tool when it comes to detection. I will share with you some tips on how to maximize your usage with antibodies.
Due to the fact that antibodies are proteins, their storage should be controlled and limited to freezers for long term storage (-20°C) or 4°C for day-to-day usage. Freeze-thaw cycles should be avoided. Regardless whether the antibody stock is commercial or self-prepared, it’s recommended that you aliquot the stock into small quantities as soon you receive it. Recommended aliquots volume should be according to your experimental requirements (try to keep them over 10ml volume) and immediately flash freeze them in liquid nitrogen. Avoid storing the antibodies at concentrations under 1mg/ml as proteins require the “crowding effect” for their stability. It is possible to dilute the antibodies stock to 50% glycerol final concentration and then keep the stocks at -20°C without passing through the sample’s freezing point. This storage should be, however, limited period time as microorganisms can grow even in -20°C.
- An exception of the above is enzyme-conjugated antibodies which should be stored at 4°C in a dark vial or wrapped in aluminum foil.
Calibrate Your Antibody
Each antibody is unique in respect to its affinity, efficacy and purity levels thus it’s important to perform a calibration of the appropriate dilution factor (or “titer”). It is recommend to first utilize a relatively simple method (western blotting or Enzyme-linked immunosorbent assay (ELISA)) to test the binding and detection of the antibody at various antibody titers. In many cases the optimal titer can be used in other application with small changes.
Chen Guttman is a Graduate Student at the Zarivach lab in Ben Gurion University. Chen blogs at benchwise and serves as BioData's community liason.