Anti-BRCA1 antibody [MS110] (ab16780)

IHC image of ab16780 staining in normal human breast formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16780, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

ICC/IF image of ab16780 stained MCF7cells. The cells were 4% PFA fixed (10 minutes) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab16780, 5 µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) used at a 1/250 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

Overlay histogram showing MCF7 cells stained with ab16780 (red line). The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab16780, 1 µg/1x10⁶ cells) for 30 minutes at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2 µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF7 cells fixed with 4% paraformaldehyde (10 minutes)/permeabilized with 0.1% PBS-Tween for 20 minutes used under the same conditions.

Immunoprecipitation step: Protein G

Sample: HeLa whole cell lysate

Total protein input: 2000 µg

Concentration: 1 µg/mg lysate

Incubation time: 2 hours at 4°C

WB antibody: Rabbit polyclonal, HRP conjugate at 1/2500 dilution

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ab16780 staining BRAC1 in human ovarian tumor cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Acetone:Methanol and blocked with a protein block, serum-free for 1 hour at 18°C. Samples were incubated with primary antibody (1/100) for 14 hours at 4°C. An Alexa Fluor^® 488-conjugated Rabbit anti-mouse IgG (H+L) polyclonal (1/400) was used as the secondary antibody.

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ab16780 staining BRCA1 in human skin tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded tissue sections). The sections were fixed in formaldehyde and subjected to heat-mediated antigen retrieval in citrate buffer (pH 6.0) prior to blocking with 2% BSA for 1 hour at 22°C. The primary antibody was diluted 1/50 and incubated with the sample for 20 hours at 4°C. A biotin-conjugated goat anti-mouse polyclonal was used as the secondary antibody, diluted 1/800. Antibody was detected by DAB staining.

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ab16780 staining BRAC1 in human colon cancer cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 3% BSA for 30 minutes at 4°C. Samples were incubated with primary antibody (1/200) for 12 hours at 4°C. An Alexa Fluor^® 488-conjugated Goat anti-mouse IgG (H+L) polyclonal (1/1000) was used as the secondary antibody.

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