RIPAB+ FXR1 - RIP VALIDATED ANTIBODY AND PRIMER SET

General description

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.
Fragile X mental retardation-related protein 1 (FXR1) and FXR2 are members of the FMR1 family sharing a very high degree of protein similarity among them. FXR1 is an RNA binding protein and is expressed in the heart, kidney, brain, and testis. Mutations in FMR1 result in the most common form of mental retardation.

Specificity

This antibody recognizes FXR1.

Immunogen

Recombinant protein corresponding to human FXR1.

Application

Immunoprecipitation from RIP lysate:
Representative lot data.
RIP lysate from HeLa cells (~2 X 10E7 cell equivalents per IP) was subjected to immunoprecipitation using 5 µg of either a normal mouse IgG, (Cat. #CS200621), or 5 µg of Anti-FXR1 antibody (Cat. # CS204403). Ten percent of the precipitated proteins (lane 1: T7, lane 2: mouse IgG, lane 4: FXR1) and HeLa whole cell lysate (lane 3) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-FXR1 antibody (Cat. # CS204403, 1:1000).
Proteins were visualized using One-Step IP-Western kit (GenScript Cat. # L00232).
Arrow indicates Aly/REF (~68 kDa) (Figure 2).
Western Blot Analysis:
Representative lot data.
HeLa cell lysate was resolved by electrophoresis, transferred to PVDF membranes and probed with anti-FXR1 (0.5 μg/mL) dilution.
Proteins were visualized using a Goat anti-mouse IgG conjugated to HRP using a chemiluminescence detection system.
Arrow indicates FXR1 (~68 kDa) (Figure 3).
Immunocytochemistry Analysis:
Representative lot data. A 1:500 dilution from a representative lot detected FXR1 in HeLa and NIH/3T3 cell lysates. Nucleus is stained with DAPI (Blue). This antibody positively stains the Cytoplasm (Figure 4).
Immunoprecipitation Analysis:
A representative lot was used in an independent laboratory in immunoprecipitation. (Siomi, M., et al. (1996). Molecular and Cellular Biology. 3825-3832).

Research Category
Epigenetics & Nuclear Function

Research Sub Category
RNA Metabolism & Binding Proteins

Developmental Signaling

This RIPAb+ FXR1 -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:
RIP Lysate prepared from Jurkat cells (~5 X 10E7 cell equivalents per IP) were subjected to immunoprecipitation using either 5 µg of a normal mouse IgG (Cat. # CS200621), or 5 µg of Anti-FXR1 antibody (Cat. # CS204403) and the Magna RIP^® RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700).
Successful immunoprecipitation of Aly/REF-associated RNA was verified by qPCR using RIP Primers JUN, (Cat. # CS203198) (Figure 1).
Please refer to the Magna RIP (Cat. # 17-700) or EZ-Magna RIP (Cat. # 17-701) protocol for experimental details.

Target description

68 kDa

Physical form

Anti-FXR1 (Mouse Monoclonal), Part # CS204403. One vial containing 50 µg of protein G purified monoclonal antibody in buffer containing 0.1 M Tris-glycine, 0.105 M NaCl, pH 7.4, 0.05% sodium azide, before the addition of 30% glycerol. Store at -20°C.
Normal Mouse IgG, Part # CS200621. One vial containing 125 µg purified mouse IgG in 125 µL storage buffer containing 0.1% sodium azide. Store at -20°C.
RIP Primers, JUN, Part # CS203198. One vial containing 75 μL of 5 μM of each primer specific for the cDNA of human JUN.
Store at -20°C.
FOR: TCG ACA TGG AGT CCC AGG A
REV: GGC GAT TCT CTC CAG CTT CC

Format: Purified

Protein G Purified

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: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.

Analysis Note

Control
Includes negative control mouse IgG antibody and control primers specific for the cDNA of human JUN.

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.