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03-199 RIPAb+™ Phospho-eIF4E (Ser209) - RIP Validated Antibody and Primer Set, rabbit monoclonal

03-199
10 assays  10 assays per set. Recommended use: ~5 μL of antibody per RIP (dependent upon biological context).
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      Overview

      Replacement Information

      Key Specifications Table

      Species ReactivityKey Applications
      H, M, RRIP, WB
      Description
      Catalogue Number03-199
      Trade Name
      • RIPAb+
      • Upstate
      DescriptionRIPAb+™ Phospho-eIF4E (Ser209) - RIP Validated Antibody and Primer Set, rabbit monoclonal
      OverviewRIPAb+ antibodies are evaluated using the RNA Binding Protein Immunoprecipitation (RIP) assay. Each RIPAb+ antibody set includes a negative control antibody to ensure specificity of the RIP reaction and is verified for the co-immunoprecipitation of RNA associated specifically with the immunoprecipitated RNA binding protein of interest. Where appropriate, the RIPAb+ set also includes quantitative RT-PCR control primers (RIP Primers) to biologically validate your IP results by successfully co-precipitating the specific RNA targets, such as messenger RNAs. The qPCR protocol and primer sequences are provided, allowing researchers to validate RIP protocols when using the antibody in their experimental context. If a target specific assay is not provided, the RIPAb+ kit is validated using an automated microfluidics-based assay by enrichment of detectable RNA over control immunoprecipitation.
      eIF4E is a eukaryotic translation initiation factor that is involved in the direction of ribosomes to the cap structure of mRNA's. There are two forms of eIF4E: a free form (25 kDa) and as part of a multi-protein complex (eIF-4F). The free form seems to not be as plentiful, compared to the other initiation factors and acts as a rate limiting step of initiation.
      Alternate Names
      • eIF-4F 25 kDa subunit
      • eukaryotic translation initiation factor 4E
      • eukaryotic translation initiation factor 4E-like 1
      • mRNA cap-binding protein
      Background InformationeIF4E is a eukaryotic translation initiation factor that is involved in the direction of ribosomes to the cap structure of mRNA's. There are two forms of eIF4E: a free form
      (25 kDa) and as part of a multi-protein complex (eIF-4F). The free form seems to not be as plentiful, compared to the other initiation factors and acts as a rate limiting step of initiation.
      References
      Product Information
      FormatUnpurified
      Control
      • Includes negative control normal rabbit IgG antibody and control primers specific for the cDNA of human ACTB.
      PresentationAnti-Phospho-eIF4E (Ser209) (Rabbit Monoclonal). One vial containing 50 μL of Rabbit Monoclonal Antibody in buffer containing 50 mM Tris-Glycine (pH 7.4), 0.15 M NaCl containing 40% Glycerol, 0.01% sodium azide and 0.05% BSA. Store at -20°C.
      Normal Rabbit IgG. One vial containing 125 µg of Rabbit IgG in 125 µL of storage buffer containing 0.05% sodium azide. Store at -20°C.
      RIP Primers, ACTB, Part # CS203211. One vial containing 75 μL of 5 μM of each primer specific for human Beta-actin (ACTB) mRNA. Store at -20°C.
      FOR: TTG TTA CAG GAA GTC CCT TGC C
      REV: ATG CTA TCA CCT CCC CTG TGT G
      Applications
      ApplicationThis RIPAb+ Phospho-eIF4E (Ser209) -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
      Key Applications
      • RNA Binding Protein Immunoprecipitation (RIP)
      • Western Blotting
      Application NotesImmunoprecipitation from RIP lysate:
      Representative lot data.
      RIP lysate from HeLa cells (~2 X 10E7 cell equivalents per IP) was subjected to immunoprecipitation using 5 µg of either a normal rabbit IgG, (Cat. # PP64B), or 5 µg of Anti-phospho-eIF4E (Ser209) antibody (Cat. # CS207318). Twenty percent of the precipitated proteins (lane 1: normal mouse IgG, lane 2: phospho-eIF4E (Ser209) and HeLa whole cell lysate were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-phospho-eIF4E (Ser209) (Cat. # CS207318, 1:1000). Proteins were visualized using One-Step™ IP-Western kit (GenScript Cat. # L00231).
      Arrow indicates phospho-eIF4E (Ser209). (Figure 2).
      Western Blot Analysis:
      Representative lot data.
      293 cell lysate was resolved by electrophoresis, transferred to PVDF membrane and probed with Anti-phospho-eIF4E (Ser209), clone EP2151Y (1:10,000 dilution); untreated (A) and treated with AP (B).
      Proteins were visualized using a Goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
      Arrow indicates phospho-eIF4E (Ser209) (~25 kDa). (Figure 3).
      Biological Information
      ImmunogenSynthetic peptide corresponding to residues surrounding phosphorylated Ser209 of human eIF4E.
      EpitopePhosphorylated Ser209
      HostRabbit
      SpecificityThis antibody recognizes eIF4E phosphorylated at Ser209.
      IsotypeIgG
      Species Reactivity
      • Human
      • Mouse
      • Rat
      Species Reactivity NoteHuman, mouse, and rat.
      Antibody TypeMonoclonal Antibody
      Entrez Gene Number
      Entrez Gene SummaryAll eukaryotic cellular mRNAs are blocked at their 5-prime ends with the 7-methylguanosine cap structure, m7GpppX (where X is any nucleotide). This structure is involved in several cellular processes including enhanced translational efficiency, splicing, mRNA stability, and RNA nuclear export. EIF4E is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs. It is a 24-kD polypeptide that exists as both a free form and as part of a multiprotein complex termed EIF4F. The EIF4E polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. The other subunits of EIF4F are a 50-kD polypeptide, termed EIF4A (see MIM 601102), that possesses ATPase and RNA helicase activities, and a 220-kD polypeptide, EIF4G (MIM 600495) (Rychlik et al., 1987 [PubMed 3469651]).
      Gene Symbol
      • CBP
      • EIF4E1
      • EIF4EL1
      • EIF4F
      • MGC111573
      • eIF-4E
      • eIF4E
      Modifications
      • Phosphorylation
      Purification MethodUnpurified
      UniProt Number
      UniProt SummaryFUNCTION: Recognizes and binds the 7-methylguanosine-containing mRNA cap during an early step in the initiation of protein synthesis and facilitates ribosome binding by inducing the unwinding of the mRNAs secondary structures.
      SUBUNIT STRUCTURE: eIF4F is a multi-subunit complex, the composition of which varies with external and internal environmental conditions. It is composed of at least EIF4A, EIF4E and EIF4G1/EIF4G3. EIF4E is also known to interact with other partners. The interaction with EIF4ENIF1 mediates the import into the nucleus. Nonphosphorylated EIF4EBP1, EIF4EBP2 and EIF4EBP3 compete with EIF4G1/EIF4G3 to interact with EIF4E; insulin stimulated MAP-kinase (MAPK1 and MAPK3) phosphorylation of EIF4EBP1 causes dissociation of the complex allowing EIF4G1/EIF4G3 to bind and consequent initiation of translation. Rapamycin can attenuate insulin stimulation, mediated by FKBPs. Interacts mutually exclusive with EIF4A1 and EIF4A2. Interacts with NGDN and PIWIL2 By similarity. Interacts with Lassa virus Z protein.
      PTM: Phosphorylation increases the ability of the protein to bind to mRNA caps and to form the eIF4F complex.
      SEQUENCE SIMILARITIES: Belongs to the eukaryotic initiation factor 4E family.
      CAUTION: Was originally thought to be phosphorylated on Ser-53 ; this was later shown to be wrong
      Molecular Weight~25 kDa
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Quality AssuranceRNA Binding Protein Immunoprecipitation:
      Representative lot data.
      RIP Lysate prepared from HeLa cells (2 X 10E7 cell equivalents per IP) were subjected to immunoprecipitation using 5 µg of either a normal rabbit IgG or 5 µg of Anti-phospho-eIF4E (Ser209) antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700).
      Successful immunoprecipitation of phospho-eIF4E (Ser209)-associated RNA was verified by qPCR using RIP Primers, ACTB, (Figure 1).
      Please refer to the Magna RIP™ (Cat. # 17-700) or EZ-Magna RIP™ (Cat. # 17-701) protocol for experimental details.protocol for experimental details.
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage ConditionsStable for 1 year at -20ºC from date of receipt.
      Handling Recommendations: Upon receipt and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.
      Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.
      Packaging Information
      Material Size10 assays
      Material Package10 assays per set. Recommended use: ~5 μL of antibody per RIP (dependent upon biological context).
      Transport Information
      Supplemental Information
      Specifications

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      Categories

      Life Science Research > Antibodies and Assays > Immunoassays > Immunoprecipitation (IP) > RNA-Binding Protein Immunoprecipitation (RIP) > RIP Validated Antibodies
      Life Science Research > Antibodies and Assays > Primary Antibodies
      Life Science Research > Protein Detection and Quantification > Immunoassays > Immunoprecipitation (IP) > RNA-Binding Protein Immunoprecipitation (RIP) > RIP Validated Antibodies