- PA1-30616 - Invitrogen Antibodies SP3 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of SP3 was achieved by transfecting HeLa with SP3 specific siRNAs (Silencer® select Product # S13324, S13325). Western Blot analysis (Fig. a) was performed using whole cell extracts from the SP3 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The Blot was probed with SP3 Polyclonal Antibody (Product # PA1-30616, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A27036, 0.25 µg/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 SP3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot was performed using SP3 Polyclonal Antibody (Product # PA1-30616) and a ~55 kDa band corresponding to SP3 was observed across cell lines and tissues tested along with an uncharacterised band (*) at ~30 kDa. Nuclear extracts (30 µg lysate) of HeLa (Lane 1), JURKAT (Lane 2), RAJI (Lane 3), U-2 OS (Lane 4) HCT-116 (Lane 5), SW480 (Lane 6), HT-29 (Lane 7), NIH/3T3 (Lane 8), PC-12 (Lane 9); and tissue extract Mouse Testis (Lane 10) and Rat testis (Lane 11) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 SP3 Polyclonal Antibody was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with SP3 Polyclonal Antibody (Product # PA1-30616) at 2 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (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 cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA1-30616