- PA5-30358 - Invitrogen Antibodies TRAP1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using TRAP1 Polyclonal Antibody (Product # PA5-30358). Sample (30 µg of whole cell lysate). Lane A: A431 . 7.5% SDS PAGE. TRAP1 Polyclonal Antibody (Product # PA5-30358) diluted at 1:3,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of TRAP1 was achieved by transfecting HeLa with TRAP1 specific siRNAs (Silencer® select Product # s177). Western blot analysis (Fig. a) was performed using membrane enriched cell extracts from the TRAP1 KD cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with TRAP1 Polyclonal Antibody (Product # PA5-30358, 1:1000 Dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/mL, 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 TRAP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using TRAP1 Rabbit Polyclonal Antibody (Product # PA5-30358) and a 74kDa band corresponding to TRAP1 was observed across cell lines and tissue extracts tested along with uncharacterized band at 110 kDa in the cell lines. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), Hep-G2 (Lane 2), HEK-293 (Lane 3), K-562 (Lane 4),T98G (Lane 5), tissue extracts (30 µg lysate) of Mouse Liver (Lane 6) and Mouse Kidney (Lane 7) were electrophoresed using NuPAGE® 4-12 % Bis-Tris 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: TRAP1 Polyclonal Antibody detects TRAP1 protein at mitochondria by immunofluorescent analysis. Sample: HeLa cells were fixed in 2% paraformaldehyde/culture medium at 37oC for 30 min and permeabilized with 100% MeOH for 30 sec. Green: TRAP1 protein stained by TRAP1 Polyclonal Antibody (Product # PA5-30358) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of TRAP1 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 TRAP1 Polyclonal Antibody (Product # PA5-30358) at 5 microgram/mL 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 cytoplasmic 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)
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Experimental details: TRAP1 Polyclonal Antibody detects TRAP1 protein at cytoplasm on human ovarian carcinoma by immunohistochemical analysis. Sample: Paraffin-embedded ovarian carcinoma. TRAP1 Polyclonal Antibody (Product # PA5-30358) dilution: 1:250. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-30358