- PA5-32428 - Invitrogen Antibodies SLC2A1 antibody

Zhou W, Cheng L, Shi Y, Ke SQ, Huang Z, Fang X, Chu CW, Xie Q, Bian XW, Rich JN, Bao S
Oncotarget 2015 Nov 10;6(35):37300-15

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of GLUT1 was achieved by transfecting MCF7 with GLUT1 specific siRNAs (Silencer® select Product # s12927, s12926). Western blot analysis (Fig. a) was performed using Membrane enriched extracts from the GLUT1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with GLUT1 Polyclonal Antibody (Product # PA5-32428, 0.5 µg/mL ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). 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 GLUT1.The target GLUT-1 is known to show a streak like pattern in positive cell lines as seen in Lanes 1 and 2. Lane 3 shows almost complete loss of streak like pick up.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-GLUT1 Polyclonal Antibody (Product # PA5-32428) and a ~42kDa band corresponding to GLUT1 was observed across cell lines and tissues tested . Membrane enriched extracts (30 µg lysate) of LNCaP (Lane 1), MCF7 (Lane 2), 3T3-L1 (Lane 3), Jurkat (Lane 4), Hep G2 (Lane 5), NIH-3T3 (Lane 6), Mouse Kidney (Lane 7), Rat Kidney (Lane 8), Mouse Liver (Lane 9) and Rat Liver (Lane 10) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). GLUT-1 shows a streak like signal in positive cell lines as seen in Lanes 1 to 6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of GLUT1 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 45 minutes at room temperature. The cells were labeled with GLUT1 Polyclonal Antibody (Product # PA5-32428) at 1:200 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), for 45 minutes at room temperature (Panel a: Blue). Nuclei (Panel b:Green) 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 Plasma membrane and cytoplasm 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 analysis of GLUT-1 using anti-GLUT-1 Polyclonal Antibody (Product # PA5-32428) in Esophagus Cancer Tissue. The recommended dilution for this antibody in immunohistochemistry applications is 1:200.

PA5-32428