- 701942 - Invitrogen Antibodies CDC42 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A431 (Lane1) and A431 treated with EGF (100 ng/mL/30 min) (2). The blots were probed with Anti-Phospho-Rac1/Cdc42pS71 Recombinant Rabbit Monoclonal Antibody (Product # 701942, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 21 kDa band corresponding to Phospho-Rac1/Cdc42pS71 was observed across cell line tested according to the given treatment. To confirm the specificity of Anti-Phospho-Rac1/Cdc42 (pS71) Recombinant Rabbit Monoclonal Antibody, competition was performed with the phosphopeptide (10 µg/mL) as shown in the corresponding blot on the right. The peptide competes with the antibody and prevents it from binding to the target protein. 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® 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: Knockout of RAC1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1044298_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of RAC1 was performed by loading 60 µg of A-431 Wild type (Lane 1), A-431 Cas9 (Lane 2) andA-431 RAC1 KO (Lane 3) modified whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Phospho-RAC1/CDC42 (Ser71) Recombinant Rabbit Monoclonal Antibody (20H14L7) (Product # 701942, 1:250 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL 1500 (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to RAC1. Uncharacterized band was observed in all the samples at ~39 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis A431 cells were fixed and permeabilized for detection of Phospho-Rac1/Cdc42 (pS71) using Phospho-Rac1/Cdc42pS71 Recombinant Rabbit Monoclonal Antibody (Product # 701942, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of Rac1/Cdc42 (pS71) (green) in the cytoplasmic can be clearly observed in cells treated with EGF (200 ng, 10 min) as compared to untreated cells. Antibody specificity was demonstrated by competition with the Rac1/Cdc4 2 (pS71) peptide, which results in loss of signal. No competition was observed with the non-phospho peptide.

701942

Antibody data