Antibodies distributed by Addgene have been produced in-house by Addgene scientists. They are generated using standard methods individually optimized for each specific antibody in order to generate high quality preparations. Following production, all antibody preps are titered and undergo stringent quality control by Addgene or a trusted quality control partner before distribution. Details about our production protocols, titering methods, and quality control are described below. Information for a specific antibody can be found on the antibody's catalog page or by contacting [email protected].
For recombinant antibodies (rAb) encoded by mammalian expression vectors, human embryonic kidney cells are transfected with the plasmid(s) encoding the heavy and light chains. Antibodies are expressed for 7 days and then purified from the supernatant using Protein A or Protein G affinity columns.
After affinity chromatography, antibodies undergo a buffer exchange using centrifugal columns. The final formulation buffer is phosphate-buffered saline containing 1 mM sodium azide. This buffer is not compatible for use in live cells and will interfere with conjugation. For applications such as these, the buffer can be removed by dialysis or gel filtration.
Titer and Purity
Antibodies are initially titered by spectrophotometry using a NanoDrop spectrophotometer. If needed, the sample is adjusted by diluting or concentrating to a final concentration of 0.9-1.1 mg/mL. Antibody concentration is confirmed by Coomassie staining.
For Coomassie staining, the sample is separated via a denaturing SDS-PAGE gel alongside serial dilutions of a standard antibody of known concentration and stained with Coomassie blue. Protein band intensities are measured using ImageJ software and a standard curve is generated. The final antibody concentration is calculated from the standard curve.
Using ImageJ software, the band intensities of both the antibody and impurities are measured and the ratio of the antibody protein content to the total protein content of the sample is determined. Only antibody samples with >90% purity are distributed.
Each lot of antibody undergoes testing, either in-house or through outside labs, to confirm recognition of the expected target or to confirm expected labeling across a tissue panel in one of the antibody’s recommended applications. The specific QC experiments performed varies for each antibody catalog item. Whenever possible, the experiment is performed in parallel with a previous lot of recombinant antibody or the hybridoma-derived equivalent to ensure that potency and specificity have been retained. If the target antigen is not endogenously-expressed (e.g., using an anti-GFP antibody), the target antigen is first transiently expressed in mammalian cells. To learn which specific QC experiments were performed on your lot, please email [email protected].
- Immunocytochemistry (ICC)
Cells are fixed and labeled with the antibody and a species-specific fluorescently-labeled secondary antibody. The fluorescence pattern is compared to an untransfected control.
- Immunohistochemistry (IHC)
Tissue sections are fixed, permeabilized, and labeled with the antibody and a species-specific horseradish peroxidase (HRP)-conjugated secondary antibody. The tissue labeling pattern is compared to the mRNA expression pattern cited in the Tissue Atlas (Link opens in a new window).
- Western Blotting (WB)
Proteins are extracted from cells, separated by SDS-PAGE, transferred to a membrane, and labeled with the antibody and a species-specific HRP-conjugated secondary antibody. Protein expression is compared to an untransfected control and the protein size is confirmed.
- Residual Plasmid Sanger Sequencing
Purified recombinant antibody preps contain low levels of co-purified plasmid from transfection. This residual plasmid is extracted and amplified with primers targeting the heavy chain and/or light chain variable regions. The PCR product is purified, sequenced via the Sanger method, and aligned with the parental plasmid sequence to confirm the antibody’s identity.
For more details on protocols see the Addgene Protocols page or the Neuromab Protocols (Link opens in a new window) page.
Storage and Shipping Stability
Addgene does not recommend repeated freeze thaw of antibodies and suggests preparing single-use aliquots before storing at -20 °C.
Each antibody catalog item is tested for shipping stability by incubating at 37 °C for 2 weeks, freezing at -80 °C, thawing on ice, and then testing for loss of potency against a non-treated control in one of its recommended assays.
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