- PA5-29674 - Invitrogen Antibodies PBX1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of PBX1 using 30 µg of A431 lysate. Samples were loaded onto a 10% SDS-PAGE gel and probed with a PBX1 polyclonal antibody (Product # PA5-29674) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of PBX1 was performed by separating 30 µg of HeLa whole cell and nuclear extracts by 10% SDS-PAGE. Proteins were transferred to a membrane and probed with a PBX1 Polyclonal Antibody (Product # PA5-29674) at a dilution of 1:1000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of PBX1 was achieved by transfecting HeLa with PBX1 specific siRNAs (Silencer® select Product # s10080, s10079). Western blot analysis (Fig. a) was performed using Whole Cell Extract-WCL from the PBX1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with PBX1 Polyclonal Antibody (Product # PA5-29674, 1:1000 dilution ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b, 50 kDa band) and (Fig c, 40 kDa band). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to PBX1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-PBX1 Polyclonal Antibody (Product # PA5-29674) and a 40 kDa and 50 kDa band corresponding to PBX1 was observed across the cell lines tested. Whole Cell Extract-WCL (30 ug µg lysate) of HEL 92.1.7 (Lane 1), IMR-32 (Lane 2), NIH:OVCAR-3 (Lane 3), HeLa (Lane 4), A-431 (Lane 5), K-562 (Lane 6) and COS-7 (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) 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)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of PBX1 was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: PBX1 Polyclonal Antibody (Product # PA5-29674) diluted at 1:500. Red: phalloidin, a cytoskeleton marker. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of PBX1 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with PBX1 Polyclonal Antibody (Product # PA5-29674) at 1:1000 dilution, in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing Nuclear and cytosolic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X with Oil immersion magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous PBX1 protein using PBX1 Antibody: ChIP was performed using Anti-PBX1 Polyclonal Antibody (Product # PA5-29674, 2.5 µg) on sheared chromatin from HeLa 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 SPOCK2 and MEOX1 (Active) and ACTB and SAT2 satellite repeats (Inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: PBX1 Polyclonal Antibody immunoprecipitates PBX1 protein in IP experiments. IP Sample: 293T whole cell lysate/extract A. Control with 2 µg of preimmune rabbit IgG B. Immunoprecipitation of PBX1 protein by 2 µg of PBX1 Polyclonal Antibody (Product # PA5-29674) 12 % SDS-PAGE The immunoprecipitated PBX1 protein was detected by PBX1 Polyclonal Antibody (Product # PA5-29674) diluted at 1:1,000. .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: PBX1 Polyclonal Antibody immunoprecipitates PBX1 protein in IP experiments. IP Sample: 293T whole cell lysate/extract A. Control with 2 µg of preimmune rabbit IgG B. Immunoprecipitation of PBX1 protein by 2 µg of PBX1 Polyclonal Antibody (Product # PA5-29674) 12 % SDS-PAGE The immunoprecipitated PBX1 protein was detected by PBX1 Polyclonal Antibody (Product # PA5-29674) diluted at 1:1,000. .

PA5-29674