PA5-17531

antibody from Invitrogen Antibodies

Targeting: IRAK4 NY-REN-64

Immunoprecipitation

Antibody data

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Validation data

IRAK4 protein structure - PA5-17531 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of IRAK4 in extracts from RAW 264.7 and H-4-II-E cell lines using IRAK4 polyclonal antibody (Product # PA5-17531).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of IRAK4 was achieved by transfecting THP-1 cells with IRAK4 specific siRNAs (Silencer® select Product # s535019, s535020). Western blot analysis (Fig. a) was performed using Whole Cell Extract from the IRAK4 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with IRAK4 Polyclonal Antibody (Product # PA5-17531, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 IRAK4, however an uncharacterized (*) band was observed at ~60 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-IRAK4 Polyclonal Antibody (Product # PA5-17531) and a 52 kDa band corresponding to IRAK4 was observed across cell lines tested. Whole Cell Extract (40 µg lysate) of THP-1 (Lane 1), THP-1 treated with PMA (25 nM for 48 hours) and LPS (100 ng/ml for 24 hours) (Lane 2), THP-1 (Lane 3), THP-1 subjected to M0 macrophage differentiation and treated with LPS (100ng/ml for 12 hours) (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (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). THP-1 differentiated macrophage upon LPS treatment are reported to show down regulation in IRAK4 levels (doi:10.1016/j.cellsig.2008.10.009).