- MA5-14585 - Invitrogen Antibodies PAX5 antibody

Scalia CR, Gendusa R, Cattoretti G
Applied immunohistochemistry & molecular morphology : AIMM 2016 Jul;24(6):436-46

Sauter JL, Ambaye AB, Mount SL
Diagnostic cytopathology 2015 Sep;43(9):688-95

Yu G, Kong L, Qu G, Zhang Q, Wang W, Jiang L
Diagnostic pathology 2011 Jul 6;6:60

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-PAX5 Monoclonal Antibody (SP34) (Product # MA5-14585) and a 45 kDa band corresponding to PAX5 was observed in Ramos, Raji and Daudi and not in Jurkat, A549, PANC-1 and K-562. Modified whole cell extracts (1% SDS) (30 µg lysate) of Ramos (Lane 1), Raji (Lane 2), Daudi (Lane 3), Jurkat (Lane 4), A549 (Lane 5), PANC-1 (Lane 6) and K-562 (Lane 7) were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:500 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036), using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of PAX5 was performed using 70% confluent log phase Ramos cells. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with PAX5 Monoclonal Antibody (SP34) (Product # MA5-14585) at 1:250 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (Product # A27034, 1:2000 dilution) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents Jurkat cells having no expression of PAX5. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Formalin-fixed, paraffin-embedded human Tonsil stained with PAX-5 using peroxidase-conjugate and DAB chromogen. Note nuclear staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Pax-5 showing positive staining in the nucleus of paraffin-treated Human B lymphoma (right) compared with a negative control in the absence of primary antibody (left). To expose target proteins, antigen retrieval method 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 a Pax-5 monoclonal antibody (Product # MA5-14585) diluted by 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Pax-5 showing positive staining in the nucleus of paraffin-treated Human tonsil tissue (right) compared with a negative control in the absence of primary antibody (left). To expose target proteins, antigen retrieval method 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 a Pax-5 monoclonal antibody (Product # MA5-14585) diluted by 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous PAX5 protein using Anti-PAX5 Antibody: ChIP was performed using Anti-PAX5 Monoclonal Antibody (Product # MA5-14585, 5 µg) on sheared chromatin from Raji cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to BACH2 and BLNK promoter and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

MA5-14585