- PA5-16282 - Invitrogen Antibodies RBX1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of ROC1 using ROC1 Polyclonal Antibody (Product # PA5-16282) on HeLa Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of ROC1 using ROC1 Polyclonal Antibody (Product # PA5-16282) on HeLa Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), MCF7 (Lane 2), Jurkat (Lane 3), HeLa (Lane 4), HEK 293 (Lane 5) and U-87 MG (Lane 6). The blots were probed with Anti-ROC1 Rabbit Polyclonal Antibody (Product # PA5-16282, 5 µg/mL) 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). A 13 kDa band corresponding to ROC1 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of ROC1 was achieved by transfecting HeLa cells with ROC1 specific siRNAs (Silencer® select Product # s19386 and s19387). Western blot analysis (Fig a) was performed using Whole cell extract from the ROC1 knock down cells (lane 1), non-specific scrambled siRNA transfected cells (lane 3) and untransfected cells (lane 2). The blots were probed with Anti-ROC1 Rabbit Polyclonal Antibody (Product # PA5-16282, 0.2 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to ROC1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of ROC1 was performed using 70 % confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ROC1 Rabbit Polyclonal antibody (PA516282) at 5 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature 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 SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear and cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of ROC1 using ROC1 Polyclonal Antibody (Product # PA5-16282) on Native Human HeLa Cells.

PA5-16282