MA5-15164

antibody from Invitrogen Antibodies

Targeting: RPS6 S6

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Recommended by provider)

Antibody Data

Product number

MA5-15164

Provider

Invitrogen Antibodies

Product name

S6 Monoclonal Antibody (E.573.4)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

It is not recommended to aliquot this antibody.

Reactivity

Human, Mouse, Rat

Host

Rabbit

Isotype

IgG

Antibody clone number

E.573.4

Vial size

100 µL

Concentration

37 µg/mL

Storage

-20°C

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

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-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with S6 Monoclonal Antibody (E.573.4) (Product # MA5-15164, 1:1000 dilution) 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 S6.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-S6 Monoclonal Antibody (E.573.4) (Product # MA5-15164) and a 31kDa band corresponding to S6 was observed across all cell lines and tissues tested. Whole cell extracts (30 µg lysate) of A-431(Lane 1), HeLa (Lane 2), Hep G2 (Lane 3), Jurkat (Lane 4), NTERA-2 cl.D1 (Lane 5), U-87 MG (Lane 6), Rat brain (Lane 7) and Mouse brain (Lane 8) 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-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).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of S6 was performed using 70% confluent log phase 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 45 minutes at room temperature. The cells were labeled with S6 Monoclonal Antibody (E.573.4) (Product # MA5-15164) 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 dilution). Panel d represents the merged image showing cytosolic, nuclear as well as nucleolar localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.