The P51-311 monoclonal antibody binds to human BMI-1 (B lymphoma Mo-MLV insertion region 1 homolog). BMI1 is a c-myc cooperating oncogene that encodes an ~45 kDa protein that is a member of the Polycomb Group (PcG) of proteins. PcG proteins are essential for the maintenance, but not initiation, of the transcriptionally repressed state of certain developmental genes. PcG proteins are a structurally diverse group of proteins with conserved functions from fly to human cells. PcG proteins form complexes and regulate the expression of genes involved in cell cycle, DNA repair and differentiation that are crucial for maintaining the self renewal of normal and cancer stem cells. Specifically, BMI-1 is a core component of PRC1 (polycomb repressive complex 1). BMI-1, via the up-regulation of hTERT and independent of c-myc, can immortalize mammary epithelial cells. BMI-1 has also been shown to repress the INK4A locus that controls the tumor suppressors p16 and p19ARF (mouse homologue of p14ARF) in mouse models. BMI-1 plays a role in maintaining the self -renewal capacities of stem cells including hematopoietic, intestinal, retinal and neural stem cells. During antibody development, the purified P51-311 monoclonal antibody was found to detect BMI-1 by Western blot analysis of cellular lysates and by indirect immunofluorescent staining and flow cytometric analysis of fixed and permeabilized cells. Western blot analysis of BMI-1 expressed by human osteosarcoma cell line. Lysate from the human U-2 OS cell line (ATCC, HTB-96™) was electrophoresed (SDS-PAGE), transferred to a membrane and then probed with Purified Mouse Anti Human-BMI-1 antibody (Cat. No. 562527) at 4.0 μg/mL (lane 1), 2.0 μg/mL. (lane 2), and 1.0 μg/mL (lane 3). BMI-1 is identified as ~45 kDa band in the U-2 OS cell lysate by Western blotting. Image analysis of BMI-1 expressed in human osteosarcoma cell line. U-2 OS cells (ATCC, HTB-96™) were fixed with BD Cytofix™ Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) and stained with Purified Mouse Anti-Human-BMI-1 antibody (2.5 μg/mL, pseudo-colored green, Cat. No. 562527) followed by Alexa Fluor® 488 Goat Anti-Mouse Ig (Life Technologies, Cat. No. A-11001). BD Pharmingen™ Hoechst 33342 solution (Cat. No. 561908, pseudo-colored blue) was used for counterstaining. The image was captured using a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software. BD™ Phosflow Perm/Wash Buffer I (Cat. No. 557885) and 0.1 % Triton™ X-100 (Sigma, Cat. No. X-100) are also suitable for permeabilization. 562527 Rev. 2 Page 1 of 2
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