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Key Specifications Table
| Species Reactivity | Key Applications |
|---|---|
| H | ICC, IP, RIP |
| Description | |
|---|---|
| Catalogue Number | 03-250 |
| Trade Name |
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| Description | RIPAb+™ Ago3 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. Ago3 (Eukaryotic translation initiation factor 2C 3) belongs to the argonaute family and contains a PAZ and a Piwi domain. Ago3 plays a role in the RNA interference (RNAi) pathway, which is important for heterochromatin formation and accurate chromosome segregation. |
| Alternate Names |
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| Background Information | Eukaryotic translation initiation factor 2C 3, Ago3, belongs to the argonaute family and contains 1 PAZ and 1 Piwi domain. Ago3 is indicated to play a role in the RNA interference (RNAi) pathway, which is important for heterochromatin formation and accurate chromosome segregation. Unlike Ago1 and Ago2, Ago3 and AG04 do not possess the ability to dissociate miRNA duplexes in a minimal in vitro system. Re-introduction of any Ago variant into embyronic (ES) cells deficient for expression of all four Ago variants will rescue miRNA silencing defects and reduce apoptosis. |
| Product Information | |
|---|---|
| Format | Purified |
| Control |
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| Presentation | Anti-Ago3 (Mouse Monoclonal). One vial containing 50 µg of 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 | Ago3 (Eukaryotic translation initiation factor 2C 3) belongs to the argonaute family & contains a PAZ & a Piwi domain. Ago3 plays a role in the RNA interference (RNAi) pathway. |
| Key Applications |
|
| 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-Ago 3 antibody (Cat. # CS207366) and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of Ago 3-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. Immunocytochemistry Analysis: Representative lot data. Confocal fluorescent analysis of HeLa cells using Anti-Ago3, clone 4B1-F6 (Red). Actin filaments have been labeled with Alexa Fluor® 488 dye-Phalloidin (Green). Nucleus is stained with DAPI (Blue). This antibody positively stains the cytoplasm. (Alexa Fluor is a registered trademark of Life Technologies®.) Immunoprecipitation Analysis: A representative lot was used by an independent laboratory in HeLa cells. (Azuma-Mukai, A., et al. (2008). Proc Natl Acad Sci USA. 105(23):7964-7969.) |
| Biological Information | |
|---|---|
| Immunogen | Recombinant protein corresponding to human Ago3 |
| Clone | 4B1-F6 |
| Concentration | Please refer to the Certificate of Analysis for the lot-specific concentration. |
| Host | Mouse |
| Specificity | This antibody has been shown to not cross react with Ago1, Ago2, or Ago4 (Azuma-Mukai A et al. (2008) Proc. Natl. Acad. Sci. USA 105: 7964-7969). |
| Isotype | IgG2aκ |
| Species Reactivity |
|
| 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, contains a PAZ domain and a PIWI domain, and may play a role in short-interfering-RNA-mediated gene silencing. This gene is located on chromosome 1 in a tandem cluster of closely related family members including argonaute 4 and eukaryotic translation initiation factor 2C, 1. Two transcript variants encoding distinct isoforms have been identified for this gene. |
| 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) and represses the translation of mRNAs which are complementary to them. Lacks endonuclease activity and does not appear to cleave target mRNAs. SUBUNIT STRUCTURE: Interacts with EIF4B, IMP8, PRMT5 and TNRC6B. SUBCELLULAR LOCATION: Cytoplasm > P-body. |
| Molecular Weight | ~96 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-Ago 3 antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of Argonaute 3-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). |