- MA5-17285 - Invitrogen Antibodies ADAR antibody

Dhillon P, Rao CD
Journal of virology 2018 Dec 15;92(24)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ADAR using A) 30 µg 293T whole cell lysate (B) 30 µg A431 whole cell lysate (C) 30 µg HeLa whole cell lysate (D) 30 µg HepG2 whole cell lysate and E) 30 µg A375 whole cell lysate. Samples were loaded onto a 5% SDS-PAGE gel and probed with an ADAR monoclonal antibody (Product # MA5-17285) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using ADAR Monoclonal Antibody (GT1066) (Product # MA5-17285). Various whole cell extracts (30 µg) were separated by 5% SDS-PAGE, and the membrane was blotted with ADAR Monoclonal Antibody (GT1066) (Product # MA5-17285) diluted at 1:1,000. The HRP-conjugated anti-mouse IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Double-stranded RNA-specific adenosine deaminase was achieved by transfecting HeLa with Double-stranded RNA-specific adenosine deaminase specific siRNAs (Silencer® select Product # S1008, S1007). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the Double-stranded RNA-specific adenosine deaminase knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ADAR Monoclonal Antibody (GT1066) (Product # MA5-17285, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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 Double-stranded RNA-specific adenosine deaminase.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-ADAR Monoclonal Antibody (GT1066) (Product # MA5-17285) and a 110 kDa band corresponding to Double-stranded RNA-specific adenosine deaminase was observed across tested cell lines along with uncharacterized bands (*) at 60 and 45 kDa. Nuclear enriched extracts (40 µg lysate) of HeLa (Lane 1), Hep G2 (Lane 2), Jurkat (Lane 3), DU 145 (Lane 4), HCT 116 (Lane 5), SH-SY5Y (Lane 6), U-87 MG (Lane 7) 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-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (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: ADAR Monoclonal Antibody (GT1066) detects ADAR1 protein at nucleus by immunofluorescent analysis. Sample: HeLa cells were fixed in 4% paraformaldehyde for 10 min. Green: ADAR1 protein stained by ADAR Monoclonal Antibody (GT1066) (Product # MA5-17285) diluted at 1:100. Red: phalloidin, a cytoskeleton marker, diluted at 1:100. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Double-stranded RNA-specific adenosine deaminase was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with ADAR Monoclonal Antibody (GT1066) (Product # MA5-17285) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). 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 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7C1115LZR).

Supportive validation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of ADAR1 was performed in 293T whole cell extracts using 5 µg of ADAR Monoclonal Antibody (GT1066) (Product # MA5-17285). Samples were transferred to a membrane and probed with ADAR Monoclonal Antibody (GT1066) as a primary antibody and an HRP-conjugated anti-Mouse IgG was used as a secondary antibody.

MA5-17285