If you are using one of our antibodies for Western Blot analysis and the reactivity seems to be diminishing over time, some points to consider are: a. Different samples were used - cells in culture will change characteristics over time, so we suggest thawing fresh cells.
b. Samples degraded in storage or from repeated freeze-thawing.
c. Antibody was contaminated - spin down vial and check for precipitate.
d. Second step reagents are not working.
e. Protein transfer was inefficient - stain blot with India Ink after transfer to confirm transfer of protein from gel to blot. Please note that antibodies are very stable and are unlikely to "go off" over time when stored appropriately.