- MA5-27777 - Invitrogen Antibodies RHEB antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of RHEB in whole cell lysate using 30 µg of protein. Samples were separated with 15% SDS-PAGE and incubated with RHEB monoclonal antibody (Product # MA5-27777) using a dilution of 1:1000 followed by HRP-conjugated anti-mouse IgG.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-RHEB Monoclonal Antibody (GT39810) (Product # MA5-27777) and a 17 kDa band corresponding to RHEB was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of PC-3 (Lane 1), DU 145 (Lane 2), LNCaP (Lane 3), HCT 116 (Lane 4), HT-29 (Lane 5), Hep G2 (Lane 6) and U-87 MG (Lane 7) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:4000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using RHEB Monoclonal Antibody (GT39810) (Product # MA5-27777). Whole cell extract (30 µg) was separated by 15% SDS-PAGE, and the membrane was blotted with RHEB Monoclonal Antibody (GT39810) (Product # MA5-27777) diluted at 1:1,000. The HRP-conjugated anti-mouse IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of RHEB was achieved by transfecting HeLa with RHEB specific siRNAs (Silencer® select Product # S12019, S12021). Western blot analysis (Fig. a) was performed using Whole cell extracts from the RHEB knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with RHEB Monoclonal Antibody (GT39810) (Product # MA5-27777, 1:1000 dilution ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:20,000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to RHEB.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of RHEB was performed in U87-MG cells fixed in 4% paraformaldehyde at RT for 15 min. Green: RHEB Monoclonal Antibody (GT39810) (Product # MA5-27777) diluted at 1:100. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of RHEB was performed using 70% confluent log phase LNCaP 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 RHEB Monoclonal Antibody (GT39810) (Product # MA5-27777) at 1:100 dilution 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 dilution), 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 merged image showing cytoplasmic 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: Immunohistochemistry (Paraffin) analysis of RHEB was performed in paraffin-embedded mouse brain tissue using RHEB Monoclonal Antibody (GT39810) (Product # MA5-27777) at a dilution of 1:200. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry (Paraffin) analysis of RHEB was performed in paraffin-embedded rat brain tissue using RHEB Monoclonal Antibody (GT39810) (Product # MA5-27777) at a dilution of 1:200. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

MA5-27777