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Key Specifications Table
| Species Reactivity | Key Applications |
|---|---|
| H | WB, RIP |
| Description | |
|---|---|
| Catalogue Number | 03-249 |
| Trade Name |
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| Description | RIPAb+™ Ago1 Antibody |
| 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. Ago1 (Eukaryotic translation initiation factor 2C 1) is a member of the argonaute family. It contains a PAZ and a PIWI domain, and is required for G1 arrest and mating in response to nitrogen starvation. It may also contribute to cell cycle control by regulating cytokinesis. Ago1 is also needed for the production of mature miRNA that affects miRNA-directed RNA cleavage and is therefore important for heterchromatin formation and accurate chromosome segregation. |
| Alternate Names |
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| Background Information | Ago1 (Eukaryotic translation initiation factor 2C 1) belongs to the argonaute family and contains 1 PAZ and 1 Piwi domain. It is required for G1 arrest and mating in response to nitrogen starvation. It is also known as a cell cycle control protein through its regulation of cytokinesis and its related cell cycle checkpoints, which occur downstream of dcr1. It is required, indirectly, for regulated hyperphosphorylation of cdc2. Ago 1 also has a role in the RNA interference (RNAi) pathway, which is important for heterochromatin formation and accurate chromosome segregation. It has been shown to be non-essential for siRNA-directed target RNA cleavage, but is needed for the production of mature miRNA that affects miRNA-directed RNA cleavage. |
| Product Information | |
|---|---|
| Format | Purified |
| Control |
|
| Presentation | Anti-Ago1 (Mouse Monoclonal). One vial containing 50 µg protein G purified monoclonal in buffer containing 0.1 M Tris-glycine (pH 7.4), 150mM NaCl, 0.05% sodium azide before the addition of glycerol to 30%. Store at -20° C. Concentration: 0.7 mg/mL Normal mouse IgG. One vial containing 125 µg purified mouse IgG in 125 µL 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 |
| Quality Level | MQ100 |
| Applications | |
|---|---|
| Application | This RIPAb+ Ago1 -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers. |
| Key Applications |
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| Application Notes | RNA Binding Protein Immunoprecipitation (microRNA): Representative lot data. RIP Lysate prepared from 293 cells (2 X 107 cell equivalents per IP) were subjected to immunoprecipitation using 5 µg of either a normal mouse IgG (Cat. # CS200621), or Anti-Ago1 antibody (Cat. # CS207365) and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of Ago 1-associated microRNA was verified by TaqMan® microRNA assay, hsa-miR-15a. (Figure2). Please refer to the Magna RIP™ (Cat. # 17-700) or EZ-Magna RIP™ (Cat. # 17-701) protocol for experimental details. Western Blot Analysis: Representative lot data. HeLa cell lysate was probed with Anti-Ago 1, clone 4G7-E12 (0.1 µg/mL). Proteins were visualized using a Goat Anti-Mouse IgG secondary antibody conjugated to HRP and a chemiluminescence detection system. Arrow indicates Ago 1 (~97 kDa). |
| Biological Information | |
|---|---|
| Immunogen | GST-tagged recombinant protein corresponding to human Ago1 |
| Clone | 4G7-E12 |
| Concentration | Please refer to the Certificate of Analysis for the lot-specific concentration. |
| Host | Mouse |
| Specificity | This antibody has been shown by an independent laboratory to not cross react with Ago2, Ago3, or Ago4 (Azuma-Mukai A et al. (2008) Proc. Natl. Acad. Sci. USA 105: 7964-7969). |
| Isotype | IgG2aκ |
| Species Reactivity |
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| Antibody Type | Monoclonal Antibody |
| Entrez Gene Number |
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| Entrez Gene Summary | 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. This gene is located on chromosome 1 in a cluster of closely related family members including argonaute 3, and argonaute 4. |
| Gene Symbol |
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| Purification Method | Protein G Purified |
| UniProt Number |
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| UniProt Summary | FUNCTION: Required for RNA-mediated gene silencing (RNAi). Binds to short RNAs such as microRNAs (miRNAs) or short interfering RNAs (siRNAs), and represses the translation of mRNAs which are complementary to them. Lacks endonuclease activity and does not appear to cleave target mRNAs. Also required for transcriptional gene silencing (TGS) of promoter regions which are complementary to bound short antigene RNAs (agRNAs). SUBUNIT STRUCTURE: Interacts with DDB1, DDX6, DDX20, DDX47, DHX9, DICER1, EIF2C2/AGO2, GEMIN4, ILF3, IMP8, MOV10, PRMT5, SART3, TARBP2, TNRC6B and UPF1. SUBCELLULAR LOCATION: Cytoplasm > P-body. |
| Molecular Weight | ~97 kDa |
| 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 Anti-Ago1 antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of Argonaute 1-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 |
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| Storage and Shipping Information | |
|---|---|
| Storage Conditions | Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon first thaw, 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 Size | 10 assays |
| Material Package | 10 assays per set. Recommended use: ~5 µg of antibody per RIP (dependent upon biological context). |