- PA5-19556 - Invitrogen Antibodies PHB antibody

Wei Y, Chiang WC, Sumpter R Jr, Mishra P, Levine B
Cell 2017 Jan 12;168(1-2):224-238.e10

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Mouse Liver Tissue Lysate using Product # PA5-19556, Prohibitin primary antibody at a dilution of 1 µg/mL (lane 1). Staining of Mouse Heart Tissue Lysate at a dilution of 1 µg/mL (lane 2). Blot treated with a secondary IR Dye680-conjugated Goat polyclonal anti-Rabbit antibody was used at a dilution of 1:10000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Prohibitin was achieved by transfecting A-431 with Prohibitin specific siRNAs (Silencer® select Product # s10425, s10424). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Prohibitin knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Prohibitin Polyclonal Antibody (Product # PA5-19556, 2 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (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 Prohibitin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Prohibitin Polyclonal Antibody(Product # PA5-19556) and a 30 kDa band corresponding to Prohibitin was observed across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of Caco-2 (Lane 1), Hep G2 (Lane 2), T-47D (Lane 3), A-431 (Lane 4), HeLa (Lane 5), NIH/3T3 (Lane 6), PC-12 (Lane 7) and tissue extracts (30 µg) of Mouse Heart (Lane 8) and Rat Brain (Lane 9) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0341BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (2 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (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 Prohibitin was performed using 60% 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 45 minutes at room temperature. The cells were labeled with Prohibitin Polyclonal Antibody (Product # PA5-19556) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000 dilution), 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 dilution). Panel d represents the merged image showing cytoplasm and nucleus 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)
Main image
Experimental details: Immunohistochemical (formalin-fixed, paraffin-embedded) staining of Human Liver tissue using Product # PA5-19510, anti-Prohibitin antibody. Primary antibody was used at a concentration of 5 µg/mL and exposed for 8 mins at room temp. The sample was pretreated using heat mediated antigen retrieval with Sodium Citrate Buffer (pH6/20mins). The detection method was a HRP conjugated polymer, DAB chromogen and the sample was counterstained with haematoxylin and mounted with DPX.

Supportive validation

Supportive validation

Supportive validation

PA5-19556