- PA5-60579 - Invitrogen Antibodies NDUFB6 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NDUFB6 in Lane 1: Marker (kDa) 250, 130, 95, 72, 55, 36, 28, 17, 10; Lane 2: Human cell line RT-4; Lane 3: Human cell line U-251MG sp; Lane 4: Human plasma (IgG/HSA depleted); Lane 5: Human liver tissue.; Lane 6: Human tonsil tissue. tissue. Samples were probed using a NDUFB6 Polyclonal Antibody (Product # PA5-60579).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of NDUFB6 was achieved by transfecting A-431 with NDUFB6 specific siRNAs (Silencer® select Product # s9364). Western blot analysis (Fig. a) was performed using whole cell extracts from the NDUFB6 KD cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with NDUFB6 Polyclonal Antibody (Product # PA5-60579, 0.4 µg/mL) 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 NDUFB6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using NDUFB6 Polyclonal Antibody (Product # PA5-60579) and a 16 kDa band corresponding to NDUFB6 was observed across cell lines and tissue extracts tested. Whole cell extracts (30 µg lysate) of A-431 (Lane 1), A549 (Lane 2), HeLa (Lane 3), U-2 OS (Lane 4), PC-3 (Lane 5), tissue extracts (30 µg lysate) of Mouse Heart (Lane 6), Mouse Liver (Lane 7), Mouse Skeletal Muscle (Lane 8) and Mouse Brain (Lane 9) were electrophoresed using NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX). 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 (0.4 µg/mL) 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: Immunofluorescent staining of NDUFB6 in human cell line A-431 shows positivity in mitochondria & nucleus but excluded from the nucleoli. Samples were probed using a NDUFB6 Polyclonal Antibody (Product # PA5-60579).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of NDUFB6 was performed using 70% confluent log phase A-431 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 NDUFB6 Rabbit Polyclonal Antibody (Product # PA5-60579) 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). Panel d represents the merged image showing Nuclear 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: Immunohistochemical staining of NDUFB6 in human prostate shows strong cytoplasmic positivity with granular pattern in glandular cells. Samples were probed using a NDUFB6 Polyclonal Antibody (Product # PA5-60579).

PA5-60579