- 701965 - Invitrogen Antibodies GLS antibody

Rossiter NJ, Huggler KS, Adelmann CH, Keys HR, Soens RW, Sabatini DM, Cantor JR
Cell metabolism 2021 Jun 1;33(6):1248-1263.e9

Kim SJ, Mehta HH, Wan J, Kuehnemann C, Chen J, Hu JF, Hoffman AR, Cohen P
Aging 2018 Jun 10;10(6):1239-1256

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on tissue extracts (30 µg lysate) of Mouse Brain (Lane 1), Rat Brain (Lane 2) and Mouse cerebellum (Lane 3). The blots were probed with Anti-Glutaminase Recombinant Rabbit Monoclonal Antibody (Product # 701965, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 66 kDa band corresponding to Glutaminase was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis was performed on fixed and permeabilized HepG2 cells for detection of endogenous Glutaminase using Anti- Glutaminase Recombinant Rabbit Monoclonal Antibody (Product # 701965, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of Glutaminase (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of Glutaminase. 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: Knockdown of GLS was achieved by transfecting Hep G2 cells with GLS specific siRNAs (Silencer® select Product # s5839, s5840). Immunofluorescence analysis was performed using untransfected Hep G2 cells (panels a, d), transfected with non-specific scrambled siRNA (panels b,e) and transfected with GLS specific siRNAs (panel c,f). Cells were fixed, permeabilized, and probed with GLS (6H5L15), Recombinant Rabbit Monoclonal Antibody (Product # 701965, 5 µg/mL), followed by labelling with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034; 1:2000 dilution) for 45 minutes at room temperature. Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962) and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Reduction of mitochondrial GLS expression was observed upon siRNA mediated knockdown (panel c,f) confirming the specificity of the antibody to GLS. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow Cytometry analysis of endogenous Glutaminase was performed on HeLa cells labeled with ABfinity™ Anti-Glutaminase Recombinant Rabbit Monoclonal Antibody (Product# 701965, 5 ug/ 1M cells) or with rabbit isotype control at 0.5 ug/ml and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product# A27034, 0.4 ug/ml, 1:2500) as represented by the red and pink histograms respectively. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (4468770).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Mitochondrial fuel usage is altered during doxorubicin-induced senescence. ( A ) Glucose uptake rate was measured by 2-NBDG, a fluorescently labeled deoxyglucose analog. Quantification and representative western blots of ( B ) carnitine palmitoyltransferase I (CPT1A) and ( C ) glutaminase (GLS1), both showing beta-actin as a loading control. Data are reported as mean +- SEM of three to six independent experiments. Significant differences were determined by Student's t -tests. *p

701965