Immunofluorescent analysis of BCR-ABL (green) in K562 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a BCR-ABL monoclonal antibody (Product # MA1-153) at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Mouse / IgG2a|
|Immunogen||Bcr686 thyroglobulin conjugate corresponding to human BCR sequence 686-696 (SSINEEITPRRQS)|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Immunofluorescence (IF)||1:50 - 1:100|
|Immunohistochemistry (Paraffin) (IHC (P))||1:50-1:500|
|Immunoprecipitation (IP)||5 µg|
|Western Blot (WB)||1:500 - 1:1000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Western blot analysis of MA1-153 detects ~130-160 kDa and a 210-250 kDa protein in K562 cells. In addition to BCR-ABL, BCR and ABL are also expressed in these cells and full length BCR is also detected by the antibody. For detection purposes, non-reducing conditions resulted in enhanced detection of BCR-ABL.
A reciprocal translocation between chromosomes 22 and 9 produces the BCR-ABL fusion oncoprotein, which is found in patients with chronic myelogenous leukemia (CML). Although there are various versions, p210 is the most commonly found BCR-ABL form. The fusion to BCR, results in deregulated/constitutively active tyrosine kinase activity of BCR-ABL which is sufficient of oncogenic transformation of myeloid cells. Inhibition of BCR-ABL oncogenic activity by the ABL-kinase inhibitor STI-571 (also known as gleevec, imatinib) became the first and best example for successful targeted therapy of cancer, and specifically CML. In recent years, several mutations (such as the T351I mutation) were found in CML patients that resulted in STI-571 resistance and the need for additional drug development.
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