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We purchased the primary antibody approximately two months ago, and it has been stored in the freezer since then. Currently, we are attempting to assess its functionality through dot blotting. In our experiment, we utilized a protein concentration of 2 μM, with a 1:1000 dilution of the primary antibody and a 1:500 dilution of the secondary antibody. The membrane was allowed to incubate in the primary antibody for 48 hours using NFDM TBST buffer. Unfortunately, the results were unsatisfactory, showing no binding, including non-specific binding. We are seeking insights into potential issues and guidance on how to determine the effectiveness of the primary antibodies.

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