- PA5-27534 - Invitrogen Antibodies HNRNPD antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using hnRNP D Polyclonal Antibody (Product # PA5-27534). Sample (30 µg of whole cell lysate). Lane A: 293T. Lane B: Raji. 10% SDS PAGE. hnRNP D Polyclonal Antibody (Product # PA5-27534) diluted at 1:10,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using hnRNP D Polyclonal Antibody (Product # PA5-27534). Sample (50 µg of whole cell lysate). Lane A: mouse brain. 10% SDS PAGE. hnRNP D Polyclonal Antibody (Product # PA5-27534) diluted at 1:5,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of hnRNP D was achieved by transfecting U-2 OS with hnRNP D specific siRNAs (Silencer® select Product # S6723, S6722). Western blot analysis (Fig. a) was performed using Whole cell extracts from the hnRNP D knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with hnRNP D Polyclonal Antibody (Product # PA5-27534, 1:8000 dilution ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). 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 D. hnRNP D isoforms observed around 42 and 48kDa showed knockdown and were quantified. The canonical isoform ~38kDa did not show knockdown.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-hnRNP D Polyclonal Antibody(Product # PA5-27534) and a ~38kDa band corresponding to hnRNP D was observed across cell lines tested. Whole cell extracts (30ug µg lysate) of HeLa (Lane 1), A549 (Lane 2), HEK-293 (Lane 3), U-2 OS (Lane 4), DU 145 (Lane 5), SH-SY5Y (Lane 6), HEL 92.1.7 (Lane 7), SK-O-V3 (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:8000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).Faint bands of other isoforms of hnRNP D were observed ~45kDa across cell lines

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of hnRNP D was performed using 70% confluent log phase U-2 OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with hnRNP D Polyclonal Antibody (Product # PA5-27534) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), for 45 minutes at room temperature (Panel a: Blue). Nuclei (Panel b:Green) 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 Nucleus and cytoplasm localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-27534