S6 pS235/pS236 Antibody, anti-human, REAfinity™

Name: S6 pS235/pS236 Antibody, anti-human, REAfinity™
Availability: InStock

S6 pS235/pS236 Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA454

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

ICFC, MICS, IF, IHC

Alternative names:

RPS6, NP33, RS6

Intracellular flow cytometry applications forS6 pS235/pS236 Antibody, anti-human, REAfinity™

APC
PE

Conjugate:

APC

Recommended dilution:

1:50

Remark:

The antibody is suited for staining of formaldehyde-fixed cells.

Tested:

QC tested

Reported:

30568034,

Products used:

130-124-302, 130-124-255

Protocols:

Intracellular flow cytometry staining protocol (Cell Signaling Buffer Set A 1:50)

Description:

Human peripheral blood mononuclear cells (PBMCs) were either left unstimulated (left peak) or stimulated with 760 nM Phorbol 12-myristate 13-acetate (PMA) at 37 °C for 15 minutes. Cells were then fixed and permeabilized using the Cell Signaling Buffer Set A followed by intracellular staining with Anti-S6 pS235/pS236 antibodies. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris were excluded from the analysis based on scatter signals.

MICS (MACSima Imaging Cyclic Staining) applications forS6 pS235/pS236 Antibody, anti-human, REAfinity™

APC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

human

Products used:

130-124-302, 130-124-255

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Extended validation for S6 pS235/pS236 Antibody, anti-human, REAfinity™

Specificity

Epitope competition

In order to compare the epitope specificity of an antibody, the clone being used is compared with other known clones recognizing the same antigen in a competition assay.

Other clones	Overlap in epitope recognition with REA454
N7-548	++
cupk43k	+
A17020B	++
Cells were incubated with an excess of purified unconjugated S6 pS235/pS236 (REA454) antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker. Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.

Sensitivity

Performance comparison

Selected fluorochrome conjugated antibodies from Miltenyi Biotec were compared to commercially available hybridoma clones in flow cytometry analysis.

Flow cytometric comparison of different clones for S6 pS235/pS236. Human peripheral blood mononuclear cells (PBMCs) were stimulated with PMA for 15min followed by a fixation and permeabilization step using the Cell Signaling Buffer Set A. Cells were then stained with S6 pS235/pS236 antibodies and plotted against the side scatter. As a control, S6 pS235/pS236 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris and cell doublets were excluded from the analysis based on scatter signals. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for S6 pS235/pS236. Human peripheral blood mononuclear cells (PBMCs) were stimulated with PMA for 15min followed by a fixation and permeabilization step using the Cell Signaling Buffer Set A. Cells were then stained with S6 pS235/pS236 antibodies and plotted against the side scatter. As a control, S6 pS235/pS236 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris and cell doublets were excluded from the analysis based on scatter signals. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for S6 pS235/pS236. Human peripheral blood mononuclear cells (PBMCs) were stimulated with PMA for 15min followed by a fixation and permeabilization step using the Cell Signaling Buffer Set A. Cells were then stained with S6 pS235/pS236 antibodies and plotted against the side scatter. As a control, S6 pS235/pS236 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris and cell doublets were excluded from the analysis based on scatter signals. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for S6 pS235/pS236. Human peripheral blood mononuclear cells (PBMCs) were stimulated with PMA for 15min followed by a fixation and permeabilization step using the Cell Signaling Buffer Set A. Cells were then stained with S6 pS235/pS236 antibodies and plotted against the side scatter. As a control, S6 pS235/pS236 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris and cell doublets were excluded from the analysis based on scatter signals. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Specifications for S6 pS235/pS236 Antibody, anti-human, REAfinity™

Overview

Clone REA454 recognizes the human 40S ribosomal protein S6 antigen phosphorylated at serine 235 and 236 (pS235/pS236). Ribosomal protein S6 is one of 33 proteins that comprise the 40 S ribosomal subunit and becomes phosphorylated at several serine residues upon mitogen stimulation. It plays an important role in controlling cell growth and proliferation through the selective translation of particular classes of mRNA. Activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for ribosomal protein S6 phosphorylation downstream of the Ras/ERK signaling cascade. The phosphorylation sites in ribosomal protein S6 have been mapped to five clustered serine residues – S235, S236, S240, S244, and S247 – which are located at the C terminus and are conserved from Drosophila to mammals. While ribosomal S6 kinase 1 phosphorylates ribosomal protein S6 at all sites, RSK exclusively phosphorylates ribosomal protein S6 at S235 and S236

in vitro

and

in vivo

using an mTOR-independent mechanism.

Additional information: Clone REA454 displays negligible binding to Fc receptors.

Alternative names

RPS6, NP33, RS6

Detailed product information

Technical specifications

Clone	REA454
Clonality	monoclonal
Isotype	recombinant human IgG1
Isotype control	REA Control Antibody (I), human IgG1
Host	human cell line
Type of antibody	Primary antibodies, Recombinant antibodies
Species	human
Antigen	S6 pS235/pS236
Alternative names of antigen	RPS6, NP33, RS6
Molecular mass of antigen [kDa]	29
Entrez Gene ID	6194
RRID	AB_2889747, AB_2889746, AB_2889745, AB_2889748

Resources for S6 pS235/pS236 Antibody, anti-human, REAfinity™

Certificates

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References for S6 pS235/pS236 Antibody, anti-human, REAfinity™

Publications

Selman, C. et al. (2009) Ribosomal protein S6 kinase 1 signaling regulates mammalian life span. Science 326(5949): 140-144
Lott, J. B. et al. (1988) Isolation and characterization of cloned cDNAs that code for human ribosomal protein S6. Gene 65(1): 31-39
Roux, P. P. et al. (2007) RAS/ERK signaling promotes site-specific ribosomal protein S6 phosphorylation via RSK and stimulates cap-dependent translation. J. Biol. Chem. 282(19): 14056-14064

Applications

Extended validation