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03-248
Sigma-AldrichRIPAb+™ pan Ago - RIP Validated Antibody and Primer Set
This RIPAb+ pan Ago -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
More>>This RIPAb+ pan Ago -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers. Less<<
SDB (Sicherheitsdatenblätter), Analysenzertifikate und Qualitätszertifikate, Dossiers, Broschüren und andere verfügbare Dokumente.
RIPAb+™ pan Ago - RIP Validated Antibody and Primer Set
Overview
RIPAb+ 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. Argonaute (Ago) proteins are a family of proteins of ~95 kDa that bind directly to mature miRNAs. In mammals, there are four Ago proteins (Ago1-4). This family of proteins is a central component of most mammalian miRNPs that mediates important functions in small RNA-directed regulatory pathways.
Alternate Names
Eukaryotic translation initiation factor 2C, 2
Protein argonaute-2
Ago2
Protein slicer
Argonaute2
PAZ Piwi domain protein
Ago4
Argonaute4
Protein argonaute-4
Background Information
Argonaute (Ago) proteins are a family of proteins of ~95 kDa that bind directly to mature miRNAs. In mammals, there are four Ago proteins (Ago1-4). This family of proteins is a central component of most mammalian miRNPs that mediates important functions in small RNA-directed regulatory pathways. The Ago family consists of two subclases, Ago and Piwi containing two characteristic domains known as PAZ and PIWI. These two domains interact with different parts of the mature miRNA. The PAZ domain interacts with the 3’-end of the miRNA, and the PIWI domain interacts with the 5’ phosphate and 5’-proximal region of the miRNA to guide target mRNA recognition.
References
Product Information
Format
Purified
Control
Includes negative control normal mouse IgG antibody and control primers specific for the cDNA of human IGF2 mRNA.
Presentation
Anti-pan Ago (Mouse Monoclonal).One vial containing 50 μg of purified mouse IgG1κ in 0.1 M Tris-Glycine (pH 7.4) and 150 mM NaCl with 0.05% sodium azide before the addition of glycerol to 30%. Store at -20°C. Normal Mouse IgG. 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 Primer IGF2. One vial containing 75 μL of 5 μM of each primer specific for human IGF2 mRNA. Store at -20°C. FOR: GCG GCT TCT ACT TCA GCA G REV: CAG GTG TCA TAT TGG AAG AAC
This RIPAb+ pan Ago -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Key Applications
Western Blotting
Immunoprecipitation
Immunocytochemistry
Immunohistochemistry
RNA Binding Protein Immunoprecipitation (RIP)
Application Notes
Western Blot Analysis: Representative lot data. HeLa cell lysate was probed with Anti-pan Ago, clone 2A8 (0.5 μg/mL). Proteins were visualized using a Goat Anti-Mouse IgG secondary antibody conjugated to HRP and chemiluminescence detection system. Arrows indicates Ago (~ 95 kDa) and Radixin (~70 kDa). (Figure 2). Immunoprecipitation Analysis: A representative lot was used by an independent laboratory in IP. (Nelson, P., et al. (2007). RNA. 13:1787–1792.) Immunocytochemistry Analysis: A representative lot was used by an independent laboratory in IC. (Nelson, P., et al. (2007). RNA. 13:1787– 1792.) Immunohistochemistry Analysis: A representative lot was used by an independent laboratory in IH. (Nelson, P., et al. (2007). RNA. 13:1787– 1792.)
Biological Information
Immunogen
Histidine - tagged recombinant protein corresponding to human Ago.
Clone
2A8
Concentration
Please refer to the Certificate of Analysis for the lot-specific concentration.
Host
Mouse
Specificity
This antibody recognizes Ago1, Ago2, Ago3, and Ago4. Note that this antibody has been reported to cross-react with radixin (~70 kDa). (Nelson, P., et al. (2007). RNA. 13:1787–1792.)
This gene encodes a member of the Argonaute family of proteins which play a role in RNA interference. The encoded protein is highly basic, and contains a PAZ domain and a PIWI domain. It may interact with dicer1 and play a role in short-interfering-RNA-mediated gene silencing. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq].
FUNCTION: Required for RNA-mediated gene silencing (RNAi) by the RNA-induced silencing complex (RISC). The 'minimal RISC' appears to include EIF2C2/AGO2 bound to a short guide RNA such as a microRNA (miRNA) or short interfering RNA (siRNA). These guide RNAs direct RISC to complementary mRNAs that are targets for RISC-mediated gene silencing. The precise mechanism of gene silencing depends on the degree of complementarity between the miRNA or siRNA and its target. Binding of RISC to a perfectly complementary mRNA generally results in silencing due to endonucleolytic cleavage of the mRNA specifically by EIF2C2/AGO2. Binding of RISC to a partially complementary mRNA results in silencing through inhibition of translation, and this is independent of endonuclease activity. May inhibit translation initiation by binding to the 7-methylguanosine cap, thereby preventing the recruitment of the translation initiation factor eIF4-E. May also inhibit translation initiation via interaction with EIF6, which itself binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The inhibition of translational initiation leads to the accumulation of the affected mRNA in cytoplasmic processing bodies (P-bodies), where mRNA degradation may subsequently occur. In some cases RISC-mediated translational repression is also observed for miRNAs that perfectly match the 3' untranslated region (3'-UTR). Can also upregulate the translation of specific mRNAs under certain growth conditions. Binds to the AU element of the 3'-UTR of the TNF (TNF-alpha) mRNA and upregulates translation under conditions of serum starvation. Also required for transcriptional gene silencing (TGS), in which short RNAs known as antigene RNAs or agRNAs direct the transcriptional repression of complementary promoter regions.
CATALYTIC ACTIVITY: Endonucleolytic cleavage to 5'-phosphomonoester.
ENZYME REGULATION: Inhibited by EDTA.
SUBUNIT STRUCTURE: Interacts with DICER1 through its Piwi domain and with TARBP2 during assembly of the RNA-induced silencing complex (RISC). Together, DICER1, EIF2C2/AGO2 and TARBP2 constitute the trimeric RISC loading complex (RLC), or micro-RNA (miRNA) loading complex (miRLC). Within the RLC/miRLC, DICER1 and TARBP2 are required to process precursor miRNAs (pre-miRNAs) to mature miRNAs and then load them onto EIF2C2/AGO2. EIF2C2/AGO2 bound to the mature miRNA constitutes the minimal RISC and may subsequently dissociate from DICER1 and TARBP2. Note however that the term RISC has also been used to describe the trimeric RLC/miRLC. The formation of RISC complexes containing siRNAs rather than miRNAs appears to occur independently of DICER1. Interacts with EIF2C1/AGO1. Also interacts with DDB1, DDX6, DDX20, DDX47, DHX9, DHX36, EIF6, FXR1, GEMIN4, ILF3, MOV10, PRMT5, P4HA1, P4HB, SART3, TNRC6A, TNRC6B and UPF1. Interacts with the P-body components DCP1A and XRN1.
SUBCELLULAR LOCATION: Cytoplasm › P-body. Nucleus. Note: Translational repression of mRNAs results in their recruitment to P-bodies. Translocation to the nucleus requires IMP8.
DOMAIN: The Piwi domain may perform RNA cleavage by a mechanism similar to that of RNase H. However while RNase H utilizes a triad of Asp-Asp-Glu (DDE) for metal ion coordination, this protein appears to utilize a triad of Asp-Asp-His (DDH).
PTM: Hydroxylated. 4-hydroxylation appears to enhance protein stability but is not required for miRNA-binding or endonuclease activity.
SEQUENCE SIMILARITIES: Belongs to the argonaute family. Ago subfamily.
Contains 1 PAZ domain.
Contains 1 Piwi domain.
BIOPHYSIOCHEMICAL PROPERTIES: Kinetic parameters:
KM=1.1 nM for a synthetic 21-nucleotide single-stranded RNA Ref.7 Ref.18
SEQUENCE CAUTION: The sequence AAH07633.1 differs from that shown. Reason: Erroneous initiation.
The sequence AAL76093.1 differs from that shown. Reason: cDNA contains a duplication of an internal sequence at the 5' end.
The sequence BC125214 differs from that shown. Reason: Frameshift at position 450.
Molecular Weight
~ 95 and 70 kDa observed. This antibody recognizes Ago1, Ago2, Ago3, and Ago4. Note that this antibody has been reported to cross-react with radixin (~70 kDa). (Nelson, P., et al. (2007). RNA. 13:1787–1792.)
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Quality Assurance
RNA Binding Protein Immunoprecipitation: Representative lot data. RIP Lysate prepared from 293 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-pan Ago antibodyand the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of Argonaute proteins-associated RNA was verified by qPCR using RIP Primers IGF2, (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 Conditions
Stable 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 Size
10 assays
Material Package
10 assays per set. Recommended use: ~5 μg of antibody per RIP (dependent upon biological context).
Transport Information
Supplemental Information
Specifications
Global Trade Item Number
Bestellnummer
GTIN
03-248
04053252756535
Documentation
RIPAb+™ pan Ago - RIP Validated Antibody and Primer Set SDB
The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation-gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that approximately 40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.
Duplex RNAs complementary to messenger RNA inhibit translation in mammalian cells by RNA interference (RNAi). Studies have reported that RNAs complementary to promoter DNA also inhibit gene expression. Here we show that the human homologs of Argonaute-1 (AGO1) and Argonaute-2 (AGO2) link the silencing pathways that target mRNA with pathways mediating recognition of DNA. We find that synthetic antigene RNAs (agRNAs) complementary to transcription start sites or more upstream regions of gene promoters inhibit gene transcription. This silencing occurs in the nucleus, requires high promoter activity and does not necessarily require histone modification. AGO1 and AGO2 associate with promoter DNA in cells treated with agRNAs, and inhibiting expression of AGO1 or AGO2 reverses transcriptional and post-transcriptional silencing. Our data indicate key linkages and important mechanistic distinctions between transcriptional and post-transcriptional silencing pathways in mammalian cells.
Comparative expression analysis of the MAGED genes during embryogenesis and brain development. Mathieu Bertrand,Ivo Huijbers,Patrick Chomez,Olivier De Backer Developmental dynamics : an official publication of the American Association of Anatomists
230
2004
The MAGED gene subfamily contains three genes in mouse and four in human. The MAGED1, D2, and D3 proteins are highly conserved between mouse and human, whereas paralogues are less conserved between each other. This finding suggests that each MAGED protein exerts a distinct function. To get a better insight into their physiological roles, we have analyzed their expression patterns during embryogenesis and brain development. In the mouse, Maged3 expression is restricted to the central nervous system where it was mostly detected in postmitotic neurons. Maged2 is mainly expressed in tissues of mesodermal origin. The expression pattern of Maged1 roughly summarizes that of Maged2 and Maged3; however, contrary to that of Maged3, it includes the proliferative zones of the nervous system. We observed a discrepancy between Maged1 expression levels of RNA and protein, suggesting that its expression is regulated at a posttranscriptional level during the mouse development.
