SPI-Mark™ Colloidal Gold Reagents
Controls
Controls
It is of great importance to perform controls during immunogold labeling
procedures in order to confirm the specific labeling in sections and
membranes. In addition controls will determine the causes of poor
performance of individual reagents or weakness in any part of the entire
procedure. Some are included here:
(A) Omit the first antibody. This will determine if the second gold labelled
antibody is labeling non-specifically.
(B) Replace the primary antibody with non-immune serum, preferably from the
same animal before immunisation. This will determine if the primary antibody is
specific.
(C) Replace the primary antibody with an inappropriate antibody from another
species. This will also test the specificity of the primary antibody and of the
gold conjugate.
(D) Absorb the primary antibody with its antigen (if available) by addition of
approximately 1nM/l for 1 hour before the incubation. This will also test the
specificity of the primary.
(E) Use a different gold conjugate directed towards a different animal species.
This will determine if the tissue has non specific attraction for gold particles.
(F) Use a known negative tissue.
Where possible a known positive control should also be included in the series
to demonstrate the validity of the primary antibody and of the method.
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Thursday May 17, 2012
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