Buffer for Primary Antibodies

It is suggested that new monoclonal antibodies be tested in several dilutions higher than those recommended by the vendor, using 0.05 M Tris buffer (pH 6.0 and 8.6) (Boenisch, 1999). The highest dilution and the pH at which maximal staining occurs should be determined for routine use of the new antibody. This approach frequently allows for the use of antibody dilutions much higher than those recommended by the supplier. 

Note that the use of higher concentrations of monoclonal antibodies does not improve weak staining in immunohistochemistry because of paucity of or masked antigens. 

In addition to the pH, the osmolarity of the buffer used to dilute the primary antibody tends to influence the immunoreactivity of monoclonal antibodies. The changes in the molarity of Tris buffer used for diluting monoclonal antibodies are expected to result in changes in the immunoreactivity of antibodies. It has been reported that the higher the concentration of cations (e.g., Na+) in the buffer or the higher the pH in their presence, the less the immunoreactivity of the monoclonal antibodies (Boenisch, 1999). However, polyclonal antibodies may not show such an adverse effect. 

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