- PA5-31828 - Invitrogen Antibodies KDM6A antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of KDM6A using A) 30 µg U87-MG whole cell lysate (B) 30 µg SK-N-SH whole cell lysate (C) 30 µg IMR32 whole cell lysate and D) 30 µg SK-N-AS whole cell lysate. Samples were loaded onto a 5% SDS-PAGE gel and probed with a KDM6A polyclonal antibody (Product # PA5-31828) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of KDM6A was performed by separating 30 µg of various whole cell extracts by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a KDM6A Polyclonal Antibody (Product # PA5-31828) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of KDM6A was performed by separating 30 µg of various whole cell extracts by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a KDM6A Polyclonal Antibody (Product # PA5-31828) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using KDM6A Polyclonal Antibody (Product # PA5-31828). Various whole cell extracts (30 µg) were separated by 5% SDS-PAGE, and the membrane was blotted with KDM6A Polyclonal Antibody (Product # PA5-31828) diluted at 1:1,000. 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: Western blot was performed using Anti-KDM6A Polyclonal Antibody (Product # PA5-31828) and a 160 kDa band corresponding to KDM6A was observed across cell lines. Whole cell extracts (40 µg lysate) of MCF7 (Lane 1), SK-N-SH (Lane 2), A549 (Lane 3), RT-4 (Lane 4), THP-1 (Lane 5), HL-60 (Lane 6) and K-562 (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 at a 0.63 µg/mL and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of KDM6A was achieved by transfecting K-562 with KDM6A specific siRNAs (Silencer® select Product # S531230, S531232). Western blot analysis (Fig. a) was performed using Whole cell extracts from the KDM6A knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with KDM6A Polyclonal Antibody (Product # PA5-31828, 0.63 µg/mL ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20000). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to KDM6A.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of UTX/KDM6A was performed by loading 2 µg of molecular weight marker (MWM), 40 µg of ARD (a KDM6A negative cell line) or 40 µg of ARP-1 (a matched KDM6A positive cell line) cell lysate onto a NuPAGE 4-12% Bis-Tris Gel (Product # NP0321BOX) and running with Novex NuPAGE MES SDS Running buffer (Product # NP0002). Proteins were transferred to Immobulon-FL PVDF membrane and blocked for 1 hour in LI-COR blocking buffer. Membrane was incubated overnight at 4°C in LI-COR blocking buffer containing a 1:1000 dilution of KDM6A polyclonal antibody (Product # PA5-31828), followed by a 1 hour room temperature incubation in TBST containing a 1:10000 dilution of 680 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 680 (Product # A32734). Detection of KDM6A at approximately 150 kDa (indicated by the arrow) was performed using a LI-COR Odyssey CLx using the 700nm channel. (left) red channel image (right) inverted grey scale image. Data courtesy of Dr. Jonathan D. Licht’s laboratory.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of KDM6A in paraformaldehyde-fixed MCF-7 cells using a KDM6A polyclonal antibody (Product # PA5-31828) at a 1:500 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of KDM6A was performed using 70% confluent log phase K-562 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 1 hour at room temperature. The cells were labeled with KDM6A Polyclonal Antibody (Product # PA5-31828) at 1:100 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), 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). 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: Immunohistochemistry (Paraffin) analysis of KDM6A was performed in paraffin-embedded mouse spleen tissue using KDM6A Polyclonal Antibody (Product # PA5-31828) at a dilution of 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry (Paraffin) analysis of KDM6A was performed in paraffin-embedded mouse lymph node tissue using KDM6A Polyclonal Antibody (Product # PA5-31828) at a dilution of 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded H441 xenograft, using KDM6A (Product # PA5-31828) antibody at 1:250 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KDM6A Polyclonal Antibody detects KDM6A protein at nucleus on mouse duodenum by immunohistochemical analysis. Sample: Paraffin-embedded mouse duodenum. KDM6A Polyclonal Antibody (Product # PA5-31828) dilution: 1:1,000. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Cross-linked ChIP was performed with HeLa chromatin extract and 5 µg of either control rabbit IgG or a KDM6A polyclonal antibody (Product # PA5-31828). The precipitated DNA was detected by PCR with primer set targeting to RBL1 promoter.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: ChIP assay analysis of KDM6A in HeLa chromatin extracts using 5 µg of either normal rabbit IgG or KDM6A Polyclonal Antibody (Product # PA5-31828). The precipitated DNA was detected by PCR with primer set targeting to RBL1 promoter.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KDM6A Polyclonal Antibody immunoprecipitates KDM6A protein in IP experiments. IP samples: SK-N-SH whole cell extract. A. 30 µg SK-N-SH whole cell extract. B. Control with 4 µg of preimmune Rabbit IgG. C. Immunoprecipitation of KDM6A protein by 4 µg KDM6A Polyclonal Antibody (Product # PA5-31828). 5 % SDS-PAGE. The immunoprecipitated KDM6A protein was detected by KDM6A Polyclonal Antibody (Product # PA5-31828) diluted at 1:500.

PA5-31828

Antibody data