PA1-25075

antibody from Invitrogen Antibodies

Targeting: AFDN AF-6, AF6, MLLT4

Antibody data

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Validation data

Product number: PA1-25075 - Provider product page
Provider: Invitrogen Antibodies
Product name: l-Afadin Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: Recommended positive controls: MDCK, HepG2, mouse liver, rat brain.
Concentration: 1.2 mg/mL

AFDN protein structure - PA1-25075 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Afadin was achieved by transfecting Hep G2 with Afadin specific siRNAs (Silencer® select Product # S8829, S8830). Western blot analysis (Fig. a) was performed using whole cell extracts from the Afadin knockdown cells (Lane 2) and non-specific scrambled siRNA transfected cells (Lane 1). The blot was probed with Afadin Polyclonal Antibody (Product # PA1-25075, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25ug/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 Afadin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Afadin Polyclonal Antibody (Product # PA1-25075) and 80, 206 kDa bands corresponding to Afadin was observed across cell lines tested. Whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), HEK-293 (Lane 2), U-2 OS (Lane 3) and SK-OV-3 (Lane 4) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # LC2001) by XCell SureLock™ Mini-Cell and XCell II™ Blot Module (Product # EI0002). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). An uncharacterized band at ~62 kDa was also observed in the samples tested.