Western blot analysis of BCR-ABL and BCR was performed following immunoprecipitation from K562 cell lysates using a phosphotyrosine monoclonal antibody for immune complex capture, and ab187831 for western blot detection. Immunoprecipitated proteins from 500 µg whole cell lysate and 10 µl of protein ladder were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for 1 hour. The membrane was probed with ab187831 at a dilution of 1/1000 overnight rotating at 4°C, washed in TBST, and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1/20,000 for 1 hour. Chemiluminescent detection was performed.

Note: Best results were obtained using non-reducing LDS sample buffer in SDS-polyacrylamide gel electrophoresis.

Immunohistochemistry analysis of Bcr-abl showing staining in the nucleus of paraffin-embedded human hepatocarcinoma (right) compared with 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 ab187831 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.

Immunohistochemistry analysis of Bcr-abl showing staining in the nucleus of paraffin-embedded human breast carcinoma (right) compared with 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 ab187831 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.

Immunohistochemistry analysis of Bcr-abl showing staining in the nucleus and cytoplasm of paraffin-embedded mouse liver tissue (right) compared with 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 ab187831 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.