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  • 03-248 - RIPAb+ pan Ago - RIP Validated Antibody and Primer Set

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03-248 Sigma-Aldrich

RIPAb+ pan Ago - RIP Validated Antibody and Primer Set

clone 2A8, from mouse

Synonym: Eukaryotic translation initiation factor 2C, 2, Protein argonaute-2, Ago2, Protein slicer, Argonaute2, PAZ Piwi domain protein, Ago4, Argonaute4, Protein argonaute-4

  •  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   2A8, monoclonal
species reactivity   human
mfr. no.   RIPAb+
  Upstate®
application(s)   RIP: suitable
  immunocytochemistry: suitable
  immunohistochemistry: suitable
  immunoprecipitation (IP): suitable
  western blot: suitable
isotype   IgG1κ
NCBI accession no.   NP_001158095
UniProt accession no.   Q9UKV8
shipped in   dry ice

Description

General description

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.

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.

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.)

Immunogen

Histidine - tagged recombinant protein corresponding to human Ago.

Application

Research Category
Epigenetics & Nuclear Function

Research Sub Category
Transcription Factors

RNA Metabolism & Binding Proteins

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

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.)

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 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.

Target description

~ 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.)

Physical form

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

Format: Purified

Protein G

Storage and Stability

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.

Analysis Note

Control
Includes negative control normal mouse IgG antibody and control primers specific for the cDNA of 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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