- MA5-34711 - Invitrogen Antibodies MBD3 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MBD3 in U937 cell lysate. Samples were incubated with MBD3 monoclonal antibody (Product # MA5-34711), at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Methyl-CpG-binding protein MBD3 was achieved by transfecting MCF7 with MBD3 specific siRNAs (Silencer® select Product # S28737, S28736). Western blot analysis (Fig. a) was performed using nuclear enriched extracts from the Methyl-CpG-binding protein MBD3 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with MBD3 Recombinant Rabbit Monoclonal Antibody (JB61-47) (Product # MA5-34711, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution). 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 MBD3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using MBD3 Recombinant Rabbit Monoclonal Antibody (JB61-47) (Product # MA5-34711) and a 32 kDa band corresponding to Methyl-CpG-binding protein MBD3 was observed across the cell panel tested. Fresh nuclear enriched extracts (20 µg lysate) of A-673 (Lane 1), HEK-293 (Lane 2), MCF7 (Lane 3), A549 (Lane 4), Mouse Spleen (Lane 5), Mouse Testis (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX), 12 well. 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, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Methyl-CpG-binding protein MBD3 was performed using 70% confluent log phase MCF7 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 MBD3 Recombinant Rabbit Monoclonal Antibody (JB61-47) (Product # MA5-34711) at 1:200 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, 1:3000 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). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of MBD3 in SH-SY-5Y cells (green). Samples were fixed in paraformaldehyde and permeabilised with 0.25% Triton X100/PBS, incubated with MBD3 monoclonal antibody (Product # MA5-34711), followed by DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of MBD3 in NIH-3T3 cells (green). Samples were fixed in paraformaldehyde and permeabilised with 0.25% Triton X100/PBS, incubated with MBD3 monoclonal antibody (Product # MA5-34711), followed by DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of MBD3 in A549 cells (green). Samples were fixed in paraformaldehyde and permeabilised with 0.25% Triton X100/PBS, incubated with MBD3 monoclonal antibody (Product # MA5-34711), followed by DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of MBD3 in paraffin-embedded rat testis tissue. Samples were incubated with MBD3 monoclonal antibody (Product # MA5-34711), and followed by hematoxylin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of MBD3 in paraffin-embedded mouse brain tissue. Samples were incubated with MBD3 monoclonal antibody (Product # MA5-34711), and followed by hematoxylin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of MBD3 in paraffin-embedded human placenta tissue. Samples were incubated with MBD3 monoclonal antibody (Product # MA5-34711), and followed by hematoxylin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry of MBD3 in SH-SY-5Y cells (red) compared with an unlabelled control (cells without incubation with primary antibody; black). Samples were incubated with MBD3 monoclonal antibody (Product # MA5-34711) at a dilution of 1:100, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG.

MA5-34711