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Key Specifications Table
| Species Reactivity | Key Applications |
|---|---|
| H, B | RIP, IP, WB |
| Description | |
|---|---|
| Catalogue Number | 03-110 |
| Brand Family | Upstate |
| Trade Name |
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| Description | RIPAb+ Ago2 - 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. |
| Alternate Names |
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| Background Information | Ago2 (argonaute-2), also known as eukaryotic translation initiation factor 2C (EIF2C2), is an essential component for siRNA-directed RNA interference (RNAi) response. Ago2 is an endonuclease required for the unwinding of siRNA duplex and assembly of siRNA into RNA-induced silencing complexes (RISC). Ago2 interacts with DICER1 through its Piwi domain. This Piwi domain is thought to provide RNA cleavage activity via a mechanism similar to RNase H. Ago2 activity is necessary for embryonic development as well as RNA-mediated gene silencing (RNAi). |
| Product Information | |
|---|---|
| Format | Ascites |
| Control |
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| Presentation | Anti-Ago2 (Mouse Monoclonal IgG1κ). One vial containing 50 µL of ascites with 0.05% sodium azide. Store at -20°C. Normal Mouse IgG. One vial containing 125 μg of purified mouse IgG in 125 µL of stoage buffer containing 0.1% sodium azide. Store at -20°C. RIP Primers, FOS. One vial containing 75 μL of 5 μM of each primer specific for human FOS gene. Store at -20°C. FOR: GAG AGC TGG TAG TTA GTA GCA TGT TGA REV: AAT TCC AAT AAT GAA CCC AAT AGA TTA GTT A |
| Quality Level | MQ100 |
| Applications | |
|---|---|
| Application | This RIPAb+ Ago2 -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): RIP Lysate prepared from HeLa cells (2 X 107 cell equivalents per IP) were subjected to immunoprecipitation using 5 µL of either a normal mouse IgG or 5 µL of Anti-Ago2 antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of Ago2-associated microRNA was verified by TaqMan® microRNA assay, hsa-miR-23a. (Please see figures). Please refer to the Magna RIP™ (Cat. # 17-700) or EZ-Magna RIP™ (Cat. # 17-701) protocol for experimental details. Immunoprecipitation from RIP lysate: Representative lot data. RIP lysate from HeLa cells (2 X 106 cell equivalents per IP) was subjected to immunoprecipitation using 0.5 µL of either a normal mouse IgG or Anti-Ago2 antibody. Precipitated proteins (lane 1: mouse IgG, lane 2: anti-Ago2) and HeLa whole cell lysate (lane 3) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-Ago2 antibody (1:5,000 dilution). Proteins were visualized using goat anti-mouse secondary antibody conjugated to HRP and chemiluminescence detection. (Please see figures). |
| Biological Information | |
|---|---|
| Immunogen | KLH-conjugated, synthetic peptide containing the sequence YSGAGPALAPPAPPPPIQG. |
| Host | Mouse |
| Specificity | Recognizes Ago2. |
| Isotype | IgG1κ |
| Species Reactivity |
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| Species Reactivity Note | Human. Predicted to react with bovine Ago2 based on sequence homology. |
| 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. Multiple transcript variants encoding different isoforms have been found for this gene. |
| Gene Symbol |
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| UniProt Number |
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| UniProt Summary | FUNCTION: SwissProt: P45973 # Component of heterochromatin. Recognizes and binds histone H3 tails methylated at Lys-9, leading to epigenetic repression. Can interact with lamin B receptor (LBR). This interaction can contribute to the association of the heterochromatin with the inner nuclear membrane. Involved in the formation of functional kinetochore through interaction with MIS12 complex proteins. SIZE: 191 amino acids; 22225 Da SUBUNIT: Interacts with SUV420H1 and SUV420H2 (By similarity). Interacts directly with ATRX, CHAF1A, LBR, NIPBL, SP100, STAM2 and TRIM28 via the chromoshadow domain. Can interact directly with CBX3 via the chromoshadow domain. Interacts with histone H3 methylated at Lys-9. Interacts with MIS12 and C20orf127. SUBCELLULAR LOCATION: Nucleus. Centromere. Note=Component of centromeric and pericentromeric heterochromatin. Associates with chromosomes during mitosis. Associates specifically with chromatin during metaphase and anaphase. PTM: Phosphorylation of HP1 and LBR may be responsible for some of the alterations in chromatin organization and nuclear structure which occur at various times during the cell cycle (By similarity). Phosphorylated during interphase and possibly hyper- phosphorylated during mitosis. SIMILARITY: SwissProt: P45973 ## Contains 2 chromo domains. |
| Molecular Weight | 100 kDa |
| Product Usage Statements | |
|---|---|
| Quality Assurance | RNA Binding Protein Immunoprecipitation: RIP Lysate prepared from HeLa cells (2 X 107 cell equivalents per IP) were subjected to immunoprecipitation using 5 µL of either a normal mouse IgG or 5 µL of Anti-Ago2 antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of Ago2-associated RNA was verified by qPCR using RIP Primers FOS (Please see figures). 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 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 |
| Packaging Information | |
|---|---|
| Material Size | 10 assays |
| Material Package | 10 assays per set. Recommended use: ~5 μL of antibody per RIP (dependent upon biological context). |