- PA5-20764 - Invitrogen Antibodies CDC42 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of human brain tissue lysate using a CDC42 polyclonal antibody (Product # PA5-20764) at (A) 0.5 and (B) 1 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of CDC42 in human brain tissue lysate with Cdc42 Polyclonal Antibody (Product # PA5-20764) at (A) 0.5 and (B) 1 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of Cdc42 was achieved by transfecting A-431 with Cdc42 specific siRNAs (Silencer® select Product # s2765, s2766). Western blot analysis (Fig. a) was performed using whole cell extracts from the Cdc42 KD cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Cdc42 Polyclonal Antibody (Product # PA5-20764, 0.5µg/mL) and Goat anti-Chicken IgY (H+L) Secondary Antibody, HRP (Product # A16054, 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 Cdc42.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Cdc42 Polyclonal Antibody (Product # PA5-20764) and 21 kDa band corresponding to Cdc42 was observed across cell lines and tissues tested along with certain uncharacterized bands at ~ 80 kDa. Whole cell extracts (30 µg lysate) of THP-1 (Lane 1), U-937 (Lane 2), A-431 (Lane 3), K-562 (Lane 4), HeLa (Lane 5), Hep G2 (Lane 6), tissue extracts of Mouse Brain (Lane 7) and Rat Brain (Lane 8) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.5µg/mL) and detected by chemiluminescence Goat anti-Chicken IgY (H+L) Secondary Antibody, HRP (Product # A16054, 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 cdc42 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 1 hour at room temperature. The cells were labeled with Cdc42 Polyclonal Antibody (Product # PA5-20764) at 5 microgram/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor 488 (Product # A-11039) 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, 1:300). Panel d represents the merged image showing cytoskeletal and cytoplasmic localization. Panel e represents cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-20764