- PA5-18000 - Invitrogen Antibodies NUMB antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot staining of Mouse Brain lysate using Product # PA5-18000 at a concentration of 0.03 µg/mL, the primary antibody incubation was 1 hour and the detection method was chemiluminescence.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of NUMB was achieved by transfecting U-87 MG cells with NUMB specific siRNAs (Silencer® select Product # s16466, Product # s16465). 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 blots were probed using NUMB Polyclonal Antibody (Product # PA5-18000) and Rabbit anti-Goat IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014). 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 isoform.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-NUMB Polyclonal Antibody (Product # PA5-18000) on whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), PC-3 (Lane 2), DU 145 (Lane 3), A549 (Lane 4), SH-SY5Y (Lane 5), HeLa (Lane 6), Mouse lung (Lane 7), Mouse brain (Lane 8) and Mouse kidney (Lane 9) and NUMB major isoform 65, 53 kDa were observed. 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 (1:2000 dilution) and detected by Rabbit anti-Goat IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of NUMB was performed using 70% confluent log phase U-87 MG 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 NUMB Polyclonal Antibody (Product # PA5-18000) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Rabbit anti-Goat IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A-11078) 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 Cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-18000