PA5-31828
antibody from Invitrogen Antibodies
Targeting: KDM6A
UTX
Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [7]
- Immunocytochemistry [2]
- Immunohistochemistry [4]
- Chromatin Immunoprecipitation [2]
- Other assay [1]
Submit
Validation data
Reference
Comment
Report error
- Product number
- PA5-31828 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- KDM6A Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant protein fragment
- Description
- Recommended positive controls: SK-N-SH, IMR32, SK-N-AS. Predicted reactivity: Mouse (93%), Xenopus laevis (82%), Dog (86%), Bovine (98%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.
- Reactivity
- Human, Mouse
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 0.68 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
No comments: Submit comment
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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.
- 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.
- 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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)
- Main image
- 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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)
- Main image
- 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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)
- Main image
- 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- 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)
- Main image
- 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.