- 51-1000Z - Invitrogen Antibodies ARHGDIA 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 MCF-7 (Lane 1), HeLa (Lane 2), HL-60 (Lane 3), K-562 (Lane 4), U-937 (Lane 5), THP-1 (Lane 6) and U-87 MG (Lane 7). The blot was probed with Anti-RhoGDI Rabbit Polyclonal Antibody (Product # 51-1000Z, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 28 kDa band corresponding to RhoGDI was observed across the cell lines and tissues 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 RhoGDI was achieved by transfecting HeLa cells with RhoGDI specific validated siRNAs (Silencer® select Product # s698). Western blot analysis (Fig 1) was performed using whole cell extracts from the RhoGDI knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-RhoGDI Antibody, Rabbit polyclonal (Product # 51-1000Z, 1 µg/mL) 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 2). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to RhoGDI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of RhoGDI Polyclonal Antibody was performed using 70% confluent log phase SH-SY5Y 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 RhoGDI Rabbit Polyclonal Antibody (Product # 51-1000Z) at 2 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 (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing 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 (IP) of Rho-GDIa from HeLa cell lysates using (lane 1) Rb anti-Rho-GDIa (Product # 51-1000Z) and (lane 2) an irrelevant antibody, followed by Western blotting with Rb anti-Rho-GDIa (Product # 51-1000Z) (both lanes).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation (IP) of Rho-GDIa from HeLa cell lysates using (lane 1) Rb anti-Rho-GDIa (Product # 51-1000Z) and (lane 2) an irrelevant antibody, followed by Western blotting with Rb anti-Rho-GDIa (Product # 51-1000Z) (both lanes).

51-1000Z