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03-249 Sigma-Aldrich

RIPAb+ Ago1 Antibody

clone 4G7-E12, from mouse

Synonym: hAgo1, Argonaute1, eIF-2C 1, eIF2C 1, Q99, GERP95, DKFZp686M13167

  •  eCl@ss 32160702

  •  NACRES NA.32

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Properties

Related Categories Antibodies, Antibody Kits, Immunoprecipitation Kits More...
Quality Level   100
biological source   mouse
clone   4G7-E12, monoclonal
species reactivity   human
mfr. no.   RIPAb+
  Upstate®
application(s)   RIP: suitable
  western blot: suitable
isotype   IgG2aκ
NCBI accession no.   NP_036331
UniProt accession no.   Q9UL18
shipped in   dry ice

Description

General description

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.

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.

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).

Immunogen

GST-tagged recombinant protein corresponding to human Ago1

Application

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).

Research Category
Epigenetics & Nuclear Function

Research Sub Category
RNA Binding Protein (RBP)

This RIPAb+ Ago1 -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Packaging

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

Quality

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.

Target description

~97 kDa

Physical form

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

Format: Purified

Protein G Purified

Storage and Stability

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.

Analysis Note

Control
Includes normal mouse IgG and primers specific for human IGF2 mRNA.

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Legal Information

MAGNA RIP is a registered trademark of Merck KGaA, Darmstadt, Germany

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

Disclaimer

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.

Safety & Documentation

Safety Information

Safety Information for this product is unavailable at this time.
Protocols & Articles

Articles

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Peer-Reviewed Papers
15

References

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