- PA5-17642 - Invitrogen Antibodies GTF2I antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of GTF2I was achieved by transfecting HeLa with GTF2I specific siRNAs (Silencer® select Product # s61703, s61704). Western blot analysis (Fig. a) was performed using modified whole cell extracts (1% SDS) from the GTF2I KD cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with GTF2I Polyclonal Antibody (Product # PA5-17642, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (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 GTF2I.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-GTF2I Polyclonal Antibody (Product # PA5-17642) and 120 kDa band corresponding to GTF2I was observed across cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of HeLa (Lane 1), A549 (Lane 2), HEK-293 (Lane 3), K-562 (Lane 4), NTERA-2 cl.D1 (Lane 5), SH-SY5Y (Lane 6), A-431 (Lane 7), Jurkat (Lane 8) and SK-BR-3 (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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-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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of GTF2I was performed using 70% confluent log phase A549 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 GTF2I Polyclonal Antibody (Product # PA5-17642) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) 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 nuclear localization. Panel e represents 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: Immunohistochemical analysis of TFII-I in paraffin-embedded human lung carcinoma using a TFII-I polyclonal antibody (Product # PA5-17642).

PA5-17642