Anti-PAX5 antibody

• Flow Cyt

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Flow Cytometry - Anti-PAX5 antibody (AB183575)

Flow cytometry analysis of PAX5 (red histogram) on Ramos cells. Cells were harvested, fixed with 4% formaldehyde, permeabilized, washed with PBS, and incubated with ab183575 at a 1/50 dilution or with PBS alone (black histogram) for 1 hour at room temperature. For flow cytometry analysis, 30-minute incubation with DyLight 488 goat anti-rabbit IgG secondary antibody was performed. 30,000 cells were acquired for analysis.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PAX5 antibody (AB183575)

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 for 15 minutes at room temperature. Cells were probed with ab183575 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 at a dilution of 1/200 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight-554 Phalloidin and nuclei (blue) were stained with Hoechst 33342 dye.

• IP

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Immunoprecipitation - Anti-PAX5 antibody (AB183575)

Immunoprecipitation of PAX5 was performed using Raji whole cell lysates. Antigen-antibody complexes were formed by incubating 75 ug of lysate with 5ug of ab183575 overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul Protein A/G Agarose, washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer. Samples was resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween-20 for 1 hour. The membrane was probed with ab183575 at a dilution of 1/1000 overnight rotating at 4°C. Membranes were washed in TBST, and probed with an IP Detection Reagent at a dilution of 1/1000 for at least one hour. Chemiluminescent detection was performed.

All lanes:

Immunoprecipitation - Anti-PAX5 antibody (ab183575)

Predicted band size: 42 kDa

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• WB

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Western blot - Anti-PAX5 antibody (AB183575)

Detection : chemiluminescence.

Western blot demonstrating antibody specificity of the target accross different cell lysates. Expression of PAX5 was observed in all cell lysates tested in comparison to Jurkat and Hep G2 using ab183575 in western blot.

All lanes:

Western blot - Anti-PAX5 antibody (ab183575) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 30 µg

Lane 2:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 30 µg

Lane 3:

Ramos (Human Burkitt's lymphoma cell line) whole cell lysate at 30 µg

Lane 4:

Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 30 µg

Lane 5:

Daudi (Human Burkitt's lymphoma cell line) whole cell lysate at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate at 1/4000 dilution

Predicted band size: 42 kDa

Observed band size: ~45 kDa

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• WB

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Western blot - Anti-PAX5 antibody (AB183575)

All lanes:

Western blot - Anti-PAX5 antibody (ab183575) at 1/1000 dilution

Lane 1:

Ramos whole cell lysate at 75 µg

Lane 2:

Daudi whole cell lysate at 75 µg

Lane 3:

Raji whole cell lysate at 75 µg

Lane 4:

Jurkat whole cell lysate at 75 µg

Lane 5:

A20 whole cell lysate at 75 µg

Secondary

All lanes:

Goat anti rabbit HRP at 1/500 dilution

Predicted band size: 42 kDa

false