- 710405 - Invitrogen Antibodies RPS6 antibody

Aukrust I, Rosenberg LA, Ankerud MM, Bertelsen V, Hollås H, Saraste J, Grindheim AK, Vedeler A
FEBS open bio 2017 Feb;7(2):160-173

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of S6 was achieved by transfecting HeLa with S6 specific siRNAs (Silencer® select Product # s12270, s12272). Western blot analysis (Fig. a) was performed using whole cell extracts from the S6 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with S6 Antibody (9HCLC) Recombinant Rabbit Polyclonal (Product # 710405, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody HRP conjugate (Product # A27036, 1:4000 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig. b). Reduction of signal upon siRNA mediated knock down confirms that antibody is specific to S6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of RPS6 was performed by loading 30 µg of MCF7 (lane1), HEK-293 (lane2), Hep G2 (Lane3), Jurkat (lne4) and SK-N-SH (lane5) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk at 4°C overnight. RPS6 was detected at ~31 kDa using RPS6 Recombinant Rabbit Polyclonal Antibody (Product # 710405) at 0.5-1 µg/mL in 2.5 % skim milk for 3 hours at room temperature on a rocking platform. Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and 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: Western blot analysis of RPS6 in HeLa (with and without TNF alpha treatment), A431, HepG2, Jurkat, HEK293, MCF-7, and PC12 whole cell extracts (lanes 1-8 respectively) using a RPS6 Recombinant Rabbit Polyclonal Antibody (Product # 710405) at a dilution of 1 µg/mL. Samples were detected using chemiluminescence (ECL). Results show a band at ~31kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of RPS6 in HeLa cells using a RPS6 Recombinant Rabbit Polyclonal Antibody (Product # 710405) followed by detection using an Alexa Fluor 488-conjugated Goat anti-Rabbit secondary antibody (green) (Image A). Nuclei were stained using DAPI (Image B) and actin stained with Alexa Fluor 594 phalloidin (red) (image C). Image D is a composite image showing cytoplasmic localization of RPS6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of RPS6 was done on 70% confluent log phase U-2 OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with RPS6 Recombinant Rabbit Polyclonal Antibody (Product # 710405) at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 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 Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization and panel e is a no primary antibody control. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of S6 ribosomal protein (green) in 3T3 cells. The cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes, and blocked with 3% Blocker BSA in PBS (Product # 37525) for 15 minutes at room temperature. Cells were stained with a S6 Recombinant Rabbit Polyclonal Antibody (Product # 710405), at a dilution of 10 µg/mL in blocking buffer for at least 1 hour at room temperature, and then incubated with a Goat anti-Rabbit IgG Superclonal secondary antibody, Alexa Fluor 488 conjugate (Product # A27034) at a dilution of 1:1000 for 30 minutes at room temperature (green). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of RPS6 in Hela cells using a RPS6 Recombinant Rabbit Polyclonal Antibody (Product # 710405). Cells were fixed and permeabilized using FIX & PERM (Product # GAS-004) reagent, and detection was performed using an Alexa Fluor 488 Goat anti-Rabbit IgG (right peak) compared to an isotype control (left peak).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Whole cell lysate from HeLa cells were immunoprecipitated using Anti-RPS6 Recombinant Rabbit Monoclonal Antibody (Product # 710405) at 2 µg/IP and subsequently detected by Western blotting at 1 µg/mL. Rabbit IgG was used as a IP control.

710405