- PA5-27359 - Invitrogen Antibodies G6PD antibody

Submitted references Breast cancer stem cells rely on fermentative glycolysis and are sensitive to 2-deoxyglucose treatment.

Ciavardelli D, Rossi C, Barcaroli D, Volpe S, Consalvo A, Zucchelli M, De Cola A, Scavo E, Carollo R, D'Agostino D, Forlì F, D'Aguanno S, Todaro M, Stassi G, Di Ilio C, De Laurenzi V, Urbani A
Cell death & disease 2014 Jul 17;5(7):e1336

p63 isoforms regulate metabolism of cancer stem cells.

D'Aguanno S, Barcaroli D, Rossi C, Zucchelli M, Ciavardelli D, Cortese C, De Cola A, Volpe S, D'Agostino D, Todaro M, Stassi G, Di Ilio C, Urbani A, De Laurenzi V
Journal of proteome research 2014 Apr 4;13(4):2120-36

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of G6PD was performed by separating 30 µg of various whole cell extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a G6PD Polyclonal Antibody (Product # PA5-27359) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using G6PD Polyclonal Antibody (Product # PA5-27359). Sample (30 µg of whole cell lysate). A: HeLa S3. 7.5% SDS PAGE. G6PD Polyclonal Antibody (Product # PA5-27359) diluted at 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of G6PD was performed by separating 30 µg of various whole cell extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a G6PD Polyclonal Antibody (Product # PA5-27359) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of G6PD was achieved by transfecting HeLa cells with G6PD specific siRNAs (Silencer® select Product # s5447). Western blot analysis (Fig. a) was performed whole cell extracts from the HeLa knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with G6PD Polyclonal Antibody (Product # PA5-27359, 1:500) 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 G6PD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-G6PD Rabbit Polyclonal Antibody (Product # PA5-27359) and a 59 kDa band corresponding to G6PD was observed across cell lines tested and tissues except Mouse Testis and Mouse Heart. Whole cell extracts (30 µg lysate) of A549 (Lane 1), HCT 116 (Lane 2), SW480 (Lane 2), MCF7 (Lane 4), HeLa (Lane 5), 3T3-L1 (Lane 6), 3T3-L1 differentiated to Adipocytes (Lane 7), K-562 (Lane 8), Mouse Adipose (Lane 9), Mouse Testis (Lane 10) and Mouse Heart (Lane 11) 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 (1:500 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)
Main image
Experimental details: Immunofluorescence analysis of G6PD was performed using 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 1 hour at room temperature. The cells were labeled with G6PD Goat Polyclonal Antibody (Product # PA5-27359) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant 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 composite image showing Nuclear and Cytoplasmic localization of G6PD. 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 analysis of paraffin-embedded U87 xenograft, using G6PD (Product # PA5-27359) antibody at 1:300 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 The expression levels and activities of glycolytic enzymes are increased in BCSCs and correlate with altered energy and redox status of BCSCs. ( a ) WB showing PKM2, LDH-A, and G6PDH expression in BCSCs compared with SDACs. beta -Actin is used as a protein-loading control. A representative WB of three independent experiments is shown. ( b ) PK and ( c ) LDH, and enzyme activities evaluated in spheres (BCSCs) as compared with differentiated cells (SDACs). Bar graphs show the mean activity ( n =6) expressed as mu mol/mg Protein /min, and bars represent the S.E.M. ( d ) WB showing the expression of PKM2 and LDH-A in cells sorted for CD24 expression. beta -Actin is used as a loading control. ( e ) PK and ( f ) LDH activity in CD24 -/low cells compared with CD24 + BCSCs. Bar graphs show the mean activity ( n =6) expressed as mu mol/mg Protein /min, and bars represent the S.E.M. ( g ) G6PDH enzyme activity evaluated in spheres (BCSCs) as compared with differentiated cells (SDACs). Bar graphs show the mean activity ( n =6) expressed as mu mol/mg Protein /min, and bars represent the S.E.M. ( h ) NADP + /NADPH, ( i ) NAD + /NADH, and ( j ) ADP/ATP ratios in BCSCs compared with SDACs. Treatment with rotenone does not affect ATP production in BCSCs, whereas strongly increases the ADP/ATP ratio and inhibits mitochondrial ATP production in SDACs. No changes in the ADP/ATP ratio are observed in BCSCs and SDACs after addition of dimethylsulphoxide (DMSO) alone used as a vehicle con

PA5-27359

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