- MA1-147 - Invitrogen Antibodies RAP1A antibody

No submitted references: Submit reference

No comments: Submit comment

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Rap1 was performed by loading 25 µg of various whole cell lysate per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Rap1 monoclonal antibody (Product # MA1-147) at a dilution of 1:500 overnight at 4C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 32430) at a dilution of 1:15,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Rap1 (green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA/TBST (Product # 37525) for 15 minutes at room temperature. Cells were probed with a Rap1 monoclonal antibody (Product # MA1-147) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Rap1 showing staining in the cytoplasm and membrane of paraffin-embedded human colon carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Rap1 monoclonal antibody (Product # MA1-147) diluted in 3% BSA-PBS at a dilution of 1:100 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 Rap1 showing staining in the cytoplasm and membrane of paraffin-embedded human kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Rap1 monoclonal antibody (Product # MA1-147) diluted in 3% BSA-PBS at a dilution of 1:200 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: Immunoprecipitation of Rap1 was performed on NIH3T3 cells. Antigen-antibody complexes were formed by incubating 750 µg of NIH3T3 whole cell lysate with 2 µg of a Rap1 monoclonal antibody (Product # MA1-147) overnight on a rocking platform at 4øC. The immune complexes were captured on 50 µL Protein A/G Plus Agarose (Product # 20423), washed extensively, and eluted with 5X Lane Marker Reducing Sample BUffer (Product # 39000). Eluted sample (right lane) and 25 µg of NIH3T3 control lysate (left lane) were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with a Rap1 monoclonal antibody (Product # MA1-147) at a dilution of 1:500 overnight rotating at 4øC, washed in TBST, and probed with Clean-Blot IP Detection Reagent (Product # 21230) at a dilution of 1:2500 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

MA1-147