Aurion’s Bovine Serum Albumin is obtained from healthy livestock. BSA should be dissolved in an appropriate buffer, such as phosphate buffered saline, taking care not to denature the protein by foaming. The addition of BSA may cause a drop in pH of the final solution and correction may be required. As a preservative the use of NaN3 or Kathon CG is recommended.
The use of Cold Water Fish Skin Gelatin to prevent background reactions has been recommended by e.g. Behnke et al. (J. Cell Biol. 41, , 386). The product is supplied as a liquid concentrate (40%).
Tween-20™ is a non-ionic detergent with a molecular weight of about 600 and a critical micelle concentration (CMC) of 0.06-0.07% in water at room temperature. Its working mechanism may in part be based on its action as a detergent, binding to the hydrophobic moieties of water insoluble compounds, rendering them hydrophilic. In addition, immuno-compounds may become incorporated into micelles when the Tween-20™ concentration is higher than the CMC, for instance at 0.1 % in PBS at pH 7.4.
Normal sera are used to counteract the non-specific interaction between the sample and immunoglobulins. They can be added to the blocking solution and the incubation media.
As a rule the normal serum species should be the same as the secondary antibody species (e.g. use normal goat serum with goat-anti-rabbit conjugates).
Note: normal sera should not be used in combination with Protein A and Protein G gold reagents.