Background: BARD1 and Its Biological Role

BARD1 (BRCA1-Associated Ring Domain Protein 1) is an essential tumor-suppressor protein that forms a stable heterodimer with BRCA1 through their respective RING finger domains. This BRCA1–BARD1 complex exhibits E3 ubiquitin ligase activity and plays a critical role in the DNA damage response, particularly in homologous recombination repair of double-strand breaks. BARD1 is also involved in apoptosis regulation, RNA processing, and maintenance of genome stability.

Dysregulation or mutation of BARD1 has been associated with increased susceptibility to breast, ovarian, and other cancers, making it an important biomarker in oncology research. Reliable detection of BARD1 protein expression is therefore fundamental to studies investigating BRCA1-pathway function, DNA repair mechanisms, and cancer biology.

Reagent Preparation and Storage

Formulation: BARD1 Mouse mAb (Ab351164, absea #KC-30220) is supplied in PBS only, without carrier proteins such as BSA or glycerol. This formulation is suitable for applications requiring low background and is compatible with direct conjugation workflows.

Storage Conditions:

• Store the antibody at -20°C upon receipt for long-term storage.
• For short-term use (up to 1 month), the antibody may be kept at 4°C.
• Avoid repeated freeze-thaw cycles, which can reduce antibody activity. Aliquot the stock into single-use volumes before freezing.
• Do not store in a frost-free freezer.

Working Concentration: Optimal working dilutions should be determined empirically by the end user for each specific application and sample type. Refer to the datasheet for suggested starting dilution ranges.

Note: Specific dilution recommendations and lot-specific concentration data should be verified against the current product datasheet supplied with the antibody.

Step-by-Step Application Protocol

The following general protocol outlines the use of BARD1 Mouse mAb for common immunodetection applications. Users should optimize conditions for their specific experimental system.

Western Blot (WB)

1. Prepare cell or tissue lysates in an appropriate lysis buffer containing protease inhibitors.
2. Resolve 20–50 µg of total protein per lane by SDS-PAGE and transfer to a PVDF or nitrocellulose membrane.
3. Block the membrane with 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature.
4. Incubate with BARD1 Mouse mAb at the recommended dilution in blocking buffer overnight at 4°C.
5. Wash three times with TBST (10 minutes each).
6. Incubate with an appropriate HRP-conjugated secondary anti-mouse antibody for 1 hour at room temperature.
7. Wash three times with TBST and detect signal using an ECL substrate.
8. The expected molecular weight of BARD1 is approximately 90–95 kDa.

Immunofluorescence (IF)

1. Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, then permeabilize with 0.1–0.3% Triton X-100 in PBS for 10 minutes.
2. Block with 5% normal serum in PBS for 30–60 minutes.
3. Incubate with BARD1 Mouse mAb at the recommended dilution overnight at 4°C.
4. Wash three times with PBS and incubate with a fluorochrome-conjugated anti-mouse secondary antibody for 1 hour at room temperature, protected from light.
5. Counterstain nuclei with DAPI and mount coverslips.
6. Image using appropriate fluorescence channels.

Immunoprecipitation (IP)

1. Pre-clear 500 µg–1 mg of cell lysate with Protein G or Protein A beads for 1 hour at 4°C.
2. Add BARD1 Mouse mAb at the recommended amount to the pre-cleared lysate and incubate overnight at 4°C with gentle rotation.
3. Add Protein G beads and incubate for an additional 2 hours at 4°C.
4. Wash beads four times with lysis buffer, elute with SDS sample buffer, and analyze by Western blot.

Troubleshooting

The table below outlines common issues encountered when using BARD1 Mouse mAb and suggested corrective actions:

Expected Results and Quality Control

Expected Molecular Weight: BARD1 protein is expected to appear at approximately 90–95 kDa on Western blot. The observed band may differ slightly from the predicted molecular weight due to post-translational modifications.

Positive Controls: Cell lines known to express BARD1 include HeLa, MCF-7, and HEK293T. Using a positive control lysate is strongly recommended to validate antibody performance in each experiment.

Negative Controls: Omit the primary antibody and replace with an isotype-matched mouse IgG control at the same concentration to assess non-specific background signal.

Lot-to-Lot Consistency: As with all research-grade antibodies, it is advisable to retain a portion of each lot for cross-validation when switching to a new lot. Contact Aladdin Scientific technical support for lot-specific validation data.

Note: Specific validated applications and cell line expression data should be confirmed against the current Ab351164 product datasheet.