- PA5-29735 - Invitrogen Antibodies FKBP5 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of FKBP5 using 30 µg of IMR32 lysate. Samples were loaded onto a 10% SDS-PAGE gel and probed with a FKBP5 polyclonal antibody (Product # PA5-29735) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using FKBP5 Polyclonal Antibody (Product # PA5-29735). Whole cell extract (30 µg) was separated by 10% SDS-PAGE, and the membrane was blotted with FKBP5 Polyclonal Antibody (Product # PA5-29735) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of FKBP5 was achieved by transfecting HEK-293 with FKBP5 specific siRNAs (Silencer® select Product # s5214). Western blot analysis (Fig. a) was performed using whole cell extracts from the FKBP5 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with FKBP5 Polyclonal Antibody (Product # PA5-29735, 1:2000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25ug/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to FKBP5.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-FKBP5 Polyclonal Antibody (Product # PA5-29735) and both 30, 51kDa band corresponding to FKBP5 was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HT-29 (Lane 2), HCT116 (Lane 3), HEL (Lane 4), HEK-293 (Lane 5), Jurkat (Lane 6), K-562 (Lane 7), MCF-7 (Lane 8) and PC-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 (1:2000 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)
Main image
Experimental details: Immunofluorescence analysis of FKBP5 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 1% BSA for 1 hour at room temperature. The cells were labeled with FKBP5 Polyclonal Antibody (Product # PA5-29735) 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 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 and cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-29735