- MA5-15469 - Invitrogen Antibodies NANOG antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Nanog was achieved by transfecting NTERA-2 cells with Nanog specific siRNAs (Silencer® select Product # s36650). Western blot analysis (Fig. a) was performed using whole cell extracts from the NTERA-2 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and un-transfected cells (lane 1). The blots were probed with Antibody (Product # MA5-15469, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25µg/ml, 1:4000 dilution). Densitometry 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 Nanog.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on modified whole cell lysate (1% SDS) (30 µg lysate) of NCCIT (Lane 1), NTERA-2 (Lane 2) and HeLa (Lane 3) (Fig. a ), Human iPSCs (Lane 1) and Human iPSCs derived definitive endoderm (Lane 2) (Fig. b). The blot was probed with Anti- Nanog Monoclonal Antibody (Product # MA5-15469, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/ml, 1:4000 dilution). A 42 kDa (along with *160kDa a higher molecular weight) band corresponding to Nanog was observed across the cell lines tested but not in HeLa which is reported to be negative for Nanog expression and lower expression of Nanog was observed in Human iPSCs derived definitive endoderm as compared to iPSc cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NANOG using NANOG monoclonal antibody (Product # MA5-15469) in NTERA-2 cell lysate (2).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Nanog was performed using 70% confluent log phase NTERA-2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Nanog Monoclonal Antibody (Product # MA5-15469) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 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 localization. Panel e shows Nanog negative cell line HeLa with no signal. Panel f represents control cells with no primary to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of NTERA-2 cells (left) and HeLa cells (right) using NANOG monoclonal antibody (Product # MA5-15469) (Green). Red: Actin filaments have been labeled with DY-554 phalloidin.

MA5-15469