- MA5-14897 - Invitrogen Antibodies NUMB antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of DU 145 (Lane 1), PC-3 (Lane 2), U-87 MG (Lane 3), SK-OV-3 (Lane 4), MKN45 (Lane 5) and A549 (Lane 6). The blot was probed with Anti-NUMB Monoclonal Antibody (S.925.4) (Product # MA5-14897, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). 70, 65 and 55 kDa bands corresponding to NUMB was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Numb in extracts from HeLa and SK-OV-3 cells using Numb monoclonal antibody (Product # MA5-14897).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of NUMB was achieved by transfecting U- 87 MG with NUMB specific siRNAs (Silencer® select Product # s16465, s16466). Western blot analysis (Fig. a) was performed using whole cell extracts from the NUMB knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with NUMB Monoclonal Antibody (Product # MA5-14897, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/ml, 1:4000 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 NUMB.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of NUMB was performed using 70% confluent log phase LNCaP 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 NUMB Monoclonal Antibody (S.925.4) (Product # MA5-14897) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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). Panel d represents the merged image showing membrane staining. 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)
Main image
Experimental details: Immunofluorescence analysis of NUMB was performed using 70% confluent log phase DU 145 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 NUMB Monoclonal Antibody (S.925.4) (Product # MA5-14897) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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). Panel d represents the merged image showing membrane staining. 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)
Main image
Experimental details: Immunofluorescent analysis of Numb in HeLa cells using a Numb monoclonal antibody (Product # MA5-14897) (green). Actin filaments are labeled with a fluorescent red phalloidin. DNA is labeled using a fluorescent blue dye.

MA5-14897