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03-206 RIPAb+™ hnRNP U - RIP Validated Antibody and Primer Set

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03-206
10 assays  10 assays per set. Recommended use: ~5 μg of antibody per RIP (dependent upon biological context).
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      Overview

      Replacement Information

      Key Specifications Table

      Species ReactivityKey Applications
      HRIP, WB
      Description
      Catalogue Number03-206
      Trade Name
      • RIPAb+
      • Upstate
      DescriptionRIPAb+™ hnRNP U - RIP Validated Antibody and Primer Set
      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.
      Proteins of the heterogeneous nuclear ribonucleoparticles (hnRNP) family form a structurally diverse group of RNA binding proteins implicated in various functions. Recently, hnRNP proteins have been shown to hinder communication between factors bound to different splice sites. hnRNP-U, also termed scaffold attachment factor A (SAF-A), binds to pre-mRNA and nuclear matrix/scaffold attachment region DNA elements.
      Alternate Names
      • Scaffold attachment factor A
      • heterogeneous nuclear ribonucleoprotein U
      • heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A)
      • hnRNP U
      • hnRNP U protein
      • p120 nuclear protein
      Background InformationHeterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to be involved in pre-mRNA processing. However, its role in the regulation of gene expression is as yet poorly understood. Proteins of the heterogeneous nuclear ribonucleoparticles (hnRNP) family form a structurally diverse group of RNA binding proteins implicated in various functions. Recently, hnRNP proteins have been shown to hinder communication between factors bound to different splice sites. Conversely, several reports have described a positive role for some hnRNP proteins in pre-mRNA splicing. hnRNP-U, also termed scaffold attachment factor A (SAF-A), binds to pre-mRNA and nuclear matrix/scaffold attachment region DNA elements.
      References
      Product Information
      FormatPurified
      Control
      • Includes negative control normal mouse IgG antibody and control primers specific for the cDNA of human Ribosomal Protein S19.
      PresentationAnti-hnRNP U (Mouse Monoclonal), Part # CS207320. One vial containing 50 µg of protein G purified mouse IgG1 in 0.1M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide and 30% glycerol. Store at -20°C.
      Normal Mouse IgG, Part # CS200621. One vial containing 125 µg of purified mouse IgG in 125 µL of storage buffer containing 0.1% sodium azide. Store at -20°C.
      RIP Primers, Ribosomal Protein S19, Part # CS207321. One vial containing 75 μL of 5 μM of each primer specific for human c-myc 3' UTR. Store at -20°C.
      FOR: ACG CGA GCT GCT TCC ACA G
      REV: AGC TGC CAC CTG TCC GGC
      Quality LevelMQ100
      Applications
      ApplicationThis RIPAb+ hnRNP U -RIP Validated Antibody & Primer Set conveniently includes the hnRNP U 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 mouse IgG, (Cat. # CS200621), or 5 µg of Anti-hnRNP U antibody (Cat. # CS207320). ten percent of the precipitated proteins (lane 1: normal mouse IgG, lane 2: hnRNP U) and HeLa whole cell lysate (lane 3) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-hnRNP U antibody (Cat. # CS207320, 1:1000). Proteins were visualized using One-Step™ IP-Western kit (GenScript Cat. # L00231).
      Arrow indicates hnRNP U. (Figure 2).
      Automated Microfluidics-based Electrophoretic RNA Separation and Analysis (MFE):
      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 1. normal mouse IgG (Cat. # CS200621), or 2. Anti-hnRNP U antibody (Cat. # CS207320) and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700).
      Successful immunoprecipitation of hnRNP U-associated RNA was verified by automated microfluidics-based electrophoretic RNA separation and analysis. Please refer to the Magna RIP™ (Cat. # 17-700) or EZ-Magna RIP™ (Cat. # 17-701) protocol for experimental details. Electropherograms were generated by plotting fluorescence intensities versus migration times (Figure 3A). The virtual gel view was created from this data (Figure 3B).
      Western Blot Analysis:
      Representative lot data.
      K562 cell lysate was probed with Anti-hnRNP U, clone 3G6 (0.01 µg/mL). Proteins were visualized using a Goat Anti-Mouse IgG secondary antibody conjugated to HRP and a chemiluminescence detection system.

      Arrow indicates hnRNP U (~120 kDa). (Figure 4).

      Biological Information
      ImmunogenRecombinant protein corresponding to human hnRNP U.
      EpitopeUnknown
      Clone3G6
      ConcentrationPlease refer to the Certificate of Analysis for the lot-specific concentration.
      HostMouse
      SpecificityThis antibody recognizes hnRNP U.
      IsotypeIgG2bκ
      Species Reactivity
      • Human
      Antibody TypeMonoclonal Antibody
      Entrez Gene Number
      Entrez Gene SummaryThis gene belongs to the subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). The hnRNPs are RNA binding proteins and they form complexes with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. While all of the hnRNPs are present in the nucleus, some seem to shuttle between the nucleus and the cytoplasm. The hnRNP proteins have distinct nucleic acid binding properties. The protein encoded by this gene contains a RNA binding domain and scaffold-associated region (SAR)-specific bipartite DNA-binding domain. This protein is also thought to be involved in the packaging of hnRNA into large ribonucleoprotein complexes. During apoptosis, this protein is cleaved in a caspase-dependent way. Cleavage occurs at the SALD site, resulting in a loss of DNA-binding activity and a concomitant detachment of this protein from nuclear structural sites. But this cleavage does not affect the function of the encoded protein in RNA metabolism. At least two alternatively spliced transcript variants have been identified for this gene. [provided by RefSeq].
      Gene Symbol
      • hnrnpu
      • HNRPU
      • SAF-A
      • SAFA
      • U21.1
      • hnRNPU
      • p120
      • pp120
      Purification MethodProtein G Purified
      UniProt Number
      UniProt Summary"FUNCTION: Component of the CRD-mediated complex that promotes MYC mRNA stabilization. Binds to pre-mRNA. Has high affinity for scaffold-attached region (SAR) DNA. Bind to double- and single-stranded DNA and RNA.

      SUBUNIT STRUCTURE: Identified in the spliceosome C complex, at least composed of AQR, ASCC3L1, C19orf29, CDC40, CDC5L, CRNKL1, DDX23, DDX41, DDX48, DDX5, DGCR14, DHX35, DHX38, DHX8, EFTUD2, FRG1, GPATC1, HNRNPA1, HNRNPA2B1, HNRPA3, HNRNPC, HNRPF, HNRPH1, HNRPK, HNRPM, HNRNPR, HNRNPU, KIAA1160, KIAA1604, LSM2, LSM3, MAGOH, MORG1, PABPC1, PLRG1, PNN, PPIE, PPIL1, PPIL3, PPWD1, PRPF19, PRPF4B, PRPF6, PRPF8, RALY, RBM22, RBM8A, RBMX, SART1, SF3A1, SF3A2, SF3A3, SF3B1, SF3B2, SF3B3, SFRS1, SKIV2L2, SNRPA1, SNRPB, SNRPB2, SNRPD1, SNRPD2, SNRPD3, SNRPE, SNRPF, SNRPG, SNW1, SRRM1, SRRM2, SYF2, SYNCRIP, TFIP11, THOC4, U2AF1, WDR57, XAB2 and ZCCHC8. Component of the coding region determinant (CRD)-mediated complex, composed of DHX9, HNRNPU, IGF2BP1, SYNCRIP and YBX1. Identified in a mRNP complex, at least composed of DHX9, DDX3X, ELAVL1, HNRNPU, IGF2BP1, ILF3, PABPC1, PCBP2, PTBP2, STAU1, STAU2, SYNCRIP and YBX1. Identified in a mRNP granule complex, at least composed of ACTB, ACTN4, DHX9, ERG, HNRNPA1, HNRNPA2B1, HNRNPAB, HNRNPD, HNRNPL, HNRNPR, HNRNPU, HSPA1, HSPA8, IGF2BP1, ILF2, ILF3, NCBP1, NCL, PABPC1, PABPC4, PABPN1, RPLP0, RPS3, RPS3A, RPS4X, RPS8, RPS9, SYNCRIP, TROVE2, YBX1 and untranslated mRNAs. Interacts with IGF2BP1. Ligand for CR2.

      SUBCELLULAR LOCATION: Nucleus. Cytoplasm. Cell surface. Note: Localized in cytoplasmic mRNP granules containing untranslated mRNAs. Component of ribonucleosomes. Also found associated with the cell surface.

      PTM: Extensively phosphorylated.

      Arg-733 and Arg-739 are dimethylated, probably to asymmetric dimethylarginine.

      SEQUENCE SIMILARITIES: Contains 1 B30.2/SPRY domain.

      Contains 1 SAP domain.

      SEQUENCE CAUTION: The sequence AAC19382.1 differs from that shown. Reason: Miscellaneous discrepancy. Aberrant splicing.
      Molecular WeightThe calculated molecular weight is 90 kDa. However, the protein is usually observed at ~120 kDa. (Dreyfuss, G., et al. (2002). Nat Rev Mol Cell Biol. 3(3):195-205.)
      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 mouse IgG or 5 µg of Anti-hnRNP U antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700).
      Successful immunoprecipitation of hnRNP U-associated RNA was verified by qPCR using RIP Primers Ribosomal Protein S19, (Figure 1).
      Please refer to the Magna RIP™ (Cat. # 17-700) or EZ-Magna RIP™ (Cat. # 17-701) 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 -20°C 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: Variabillity 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 μg 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