Immunofluorescent analysis of PAX5 (green) in Ramos and negative control Jurkat cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were blocked with 2% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a PAX5 polyclonal antibody (Product # PA1-109) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:200 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight-554 Phalloidin (Product # 21834) and 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||Rabbit / IgG|
|Immunogen||Purified internal fragment of human recombinant PAX5 expressed in E. coli|
|Storage buffer||PBS with 1mg/ml BSA, 30% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:50|
|Immunohistochemistry (Paraffin) (IHC (P))||1:50-1:500|
|Immunoprecipitation (IP)||2-5 µg|
|Western Blot (WB)||1:500-1:2000|
* 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 PA1-109 detects a prominent ~45 kDa protein in PAX5 expressing human Ramos, Daudi, Gaji and mouse A20 B-cell lymphoma cells. No PAX5 was detected in negative control Jurkat cells. The 45 kDa band is enriched in immunoprecipitations from Raji lysate, however additional unknown high molecular weight bands are also enriched and detected by PA1-109. PAX5 was also specifically detected by immunofluorescence in the nuclei of Ramos cells compared to negative control Jurkat cells.
PAX5 is a member of the paired box (PAX) family of transcription factors. Members of this gene family typically contain a paired box domain, an octapeptide, and a paired-type homeodomain. PAX5 is expressed during B-lymphopoiesis and plays an essential role in early B, pre-B and pro-B lymphocyte development. PAX5 is also expressed in B-cell lymphomas, in developing CNS and testis and so the
encoded protein may also play a role in neural development and spermatogenesis. The PAX5 gene is located at 9p13, which is involved in t(9;14)(p13;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype, and in derived large-cell lymphomas. The translocation brings the E-mu enhancer of the IgH gene into close proximity to the PAX5 promoter, suggesting that the deregulation of transcription of PAX5 may contribute to the pathogenesis of these lymphomas. In constrast, a tumor suppressor role for PAX5 has been suggested in hepatocellular carcinoma and overexpression of PAX5 resulted in apoptosis in multiple myeloma.
PAX5 expression was also found in a subset of breast cancer cells and has been associated with better prognosis.
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