- MA5-25081 - Invitrogen Antibodies ARHGDIA antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ARHGDIA in HEK293T cells in untransfected (Left lane) and transfected (Right lane) samples using 5 µg per lane. The samples were separated by SDS-PAGE and probed with ARHGDIA (Product # MA5-25081) monoclonal antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ARHGDIA in HepG2, HeLa, HT29, A549, COS7, Jurkat, MDCK, PC12, MCF7 cells using 35 µg per lane. Samples were probed with ARHGDIA (Product # MA5-25081) monoclonal antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of RhoGDI was performed by loading 20 µg of HEK-293T WT (lane 1) and ARHGDIA RhoGDI CRISPR KO (lane 2) cell lysates in RIPA buffer onto a 4-20% gradient polyacrylamide gel (Product # WXP42012BOX). Proteins on the blot were visualized with Ponceau staining (below immunoblot). Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. RhoGDI was detected at approximately 25 kDa using RhoGDI Monoclonal Antibody (OTI1F2) (Product # MA5-25081) at a dilution of 1:1000 in 5% BSA in TBST overnight at 4 degree celsius. The blot was probed with Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Product # 62-6520) diluted to 0.2 µg/mL in TBST with 5% milk for 1 hr at room temperature. Chemiluminescent detection was performed using ECL Western Blotting Substrate. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of RhoGDI was achieved by transfecting HeLa with RhoGDI specific siRNAs (Silencer® select Product # s698, s699). Western blot analysis (Fig. a) was performed using Whole Cell Extract-WCL from the RhoGDI knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with RhoGDI Monoclonal Antibody (OTI1F2) (Product # MA5-25081, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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 RhoGDI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-RhoGDI Monoclonal Antibody (OTI1F2)(Product # MA5-25081) and a 25kDa band corresponding to RhoGDI was observed across the cell lines tested. Whole Cell Extract-WCL (30 ug µg lysate) of HEK-293 (Lane 1), SH-SY5Y (Lane 2), U-937 (Lane 3), NIH:OVCAR-3 (Lane 4), COS-7 (Lane 5), NIH/3T3 (Lane 6), HeLa (Lane 7) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0341BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot 2Dry Blotting System (Product # IB21001) . The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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)
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Experimental details: Immunofluorescence analysis of RhoGDI was performed using 70% confluent log phase HeLa 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 45 minutes at room temperature. The cells were labeled with RhoGDI Monoclonal Antibody (OTI1F2) (Product # MA5-25081) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), 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, 1:300 dilution). 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 with Oil immersion magnification.

MA5-25081