- MA1-24632 - Invitrogen Antibodies HNRNPU antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of hnRNP-U in HeLa cell extract. Samples were separated by SDS-PAGE, blotted with (Lane 1) or without (Lane 2) a. Samples were probed with a hnRNP-U monoclonal antibody (Product # MA1-24632).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of hnRNP U was achieved by transfecting HeLa with hnRNP U specific siRNAs (Silencer® select Product # s6744, s6743). Western blot analysis (Fig. a) was performed using whole cell extracts from the hnRNP U KD cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with hnRNP U Monoclonal Antibody (3G6) (Product # MA1-24632, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25µg/mL, 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 hnRNP U..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-hnRNP U Monoclonal Antibody (3G6) (Product # MA1-24632) and a 120 kDa band corresponding to hnRNP U was observed across cell lines tested. Whole cell extracts (30 µg) of HeLa (Lane 1), Jurkat (Lane 2), NTERA-2 cl.D1 (Lane 3), K-562 (Lane 4), Hep G2 (Lane 5) and RAW 264.7 (Lane 6 ) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris 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 the primary antibody (1:1000 dilution) and detected by chemiluminescence Goat Anti-Mouse IgG Secondary Antibody, HRP conjugate (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 hnRNP U 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 hnRNP U Monoclonal Antibody (3G6) (Product # MA1-24632) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: RNA Immunoprecipitation (RIP) assay of endogenous hnRNP U protein using Anti-hnRNP U Antibody: RIP assay was performed using Anti-hnRNP U Recombinant Mouse Monoclonal Antibody (Product # MA1-24632, 5 ug) on whole cell lysate from Hep G2 cells exposed to heat shock (45 degrees for 1 hour). Normal Mouse IgG was used as a negative IP control. RNA purified by RiboPure™ RNA Purification Kit (Product # AM1924) was analyzed by RT-PCR using the Power SYBR® Green RNA-to-CT™ 1-Step Kit (Product # 4389986) with the primers pairs over RRP41, ACTB, MYC, CCNA2 mRNA and MALAT non-coding RNA. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

MA1-24632