- MA1-138 - Invitrogen Antibodies BRCA1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of BRCA-1 was done on 70% confluent log phase MCF-7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with BRCA1 (MS13) Mouse Monoclonal Antibody (Product # MA1-138) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing nuclear and cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of BRCA1 was performed on human breast carcinoma tissue. To expose target proteins, antigen retrieval was performed by microwaving tissues for 8-15 minutes in 10mM sodium citrate buffer (pH 6.0). Following antigen retrieval, endogenous peroxidases were blocked with 3% hydrogen peroxide-methanol for 15 min at room temperature. Tissue slides were washed with deionized water and PBS, and then blocked in 3% BSA-PBS for 30 min at room temperature. Tissues were probed with a BRCA1 monoclonal antibody (Product # MA1-138) diluted 1:200 in 3% BSA-PBS (right panel) or incubated with buffer alone not containing primary antibody as a negative control (left panel), overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of BRCA1 was performed on human ovary carcinoma tissue. To expose target proteins, antigen retrieval was performed by microwaving tissues for 8-15 minutes in 10mM sodium citrate buffer (pH 6.0). Following antigen retrieval, endogenous peroxidases were blocked with 3% hydrogen peroxide-methanol for 15 min at room temperature. Tissue slides were washed with deionized water and PBS, and then blocked in 3% BSA-PBS for 30 min at room temperature. Tissues were probed with a BRCA1 monoclonal antibody (Product # MA1-138) diluted 1:50 in 3% BSA-PBS (right panel) or incubated with buffer alone not containing primary antibody as a negative control (left panel), overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: RNA immunoprecipitation (RIP) western of BRAC1 was performed in K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 to 10 µL of monoclonal BRAC1 antibody (Product # MA1-138) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D) and washed extensively. They were then eluted and analyzed using the Simple Western system using the same antibody as used in immunoprecipitation at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Lane 1 is the input, lane 2 no antibody IP and lane 3 is the target specific IP. Data courtesy of the Yeo lab as part of the ENCODE project.

MA1-138