- PA5-40135 - Invitrogen Antibodies GLS antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis and shRNA knock-down of Glutaminase C in non-transfected (-) and GLS1 (GAC isoform specific) shRNA-transfected MDA-MB-231 whole cell extracts (30 µg). Samples were separated by 7.5% SDS-PAGE and the membrane was probed with a Glutaminase C polyclonal antibody (Product # PA5-40135) at a dilution of 1:5000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using Glutaminase C (GAC) Polyclonal Antibody (Product # PA5-40135). Glutaminase C (GAC) Polyclonal Antibody validation by shRNA knock-down. Non-transfected (-) and GLS1 (GAC isoform specific) shRNA-transfected HCT116 whole cell extracts (30 µg) were separated by 10% SDS-PAGE, and the membrane was blotted with Glutaminase C (GAC) Polyclonal Antibody (Product # PA5-40135) at a dilution of 1:5,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Glutaminase was achieved by transfecting ACHN with Glutaminase specific siRNAs (Silencer® select Product # s5840, s5839). Western blot analysis (Fig. a) was performed using whole cell extracts from the Glutaminase knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Glutaminase C (GAC) Polyclonal Antibody (Product # PA5-40135, 1:5000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25 µg/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 Glutaminase.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Glutaminase C (GAC) Polyclonal Antibody (Product # PA5-40135) and a 55 kDa band corresponding to Glutaminase was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of MDA-MB-231 (Lane 1), ACHN (Lane 2), SK-OV-3 (Lane 3), Hep G2 (Lane 4) and NK-92 (Lane 5) 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 (1:5000 dilution) 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: Glutaminase C antibody detects Glutaminase C protein at mitochondria by immunofluorescent analysis. Sample: A549 cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: Glutaminase C protein stained by Glutaminase C antibody (Product # PA5-40135) diluted at 1:500. Red: Histone H3K9ac (acetyl Lys9), a nucleus marker, stained by Histone H3K9ac (acetyl Lys9) antibody [GT464] (Product # MA5-31512) 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 GLS was performed using 70% confluent log phase Hep G2 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 Glutaminase C (GAC) Polyclonal Antibody (Product # PA5-40135) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790) 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 mitochondrial pattern in cytoplasm. 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: Glutaminase C antibody detects Glutaminase C protein at cytoplasm on human breast carcinoma by immunohistochemical analysis. Sample: Paraffin-embedded human breast carcinoma. Glutaminase C antibody (Product # PA5-40135) diluted at 1:500.

PA5-40135