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- 规格参数
- 包装参数
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.
Proteins of the heterogeneous nuclear ribonucleoparticles (hnRNP) family form a structurally diverse group of RNA binding proteins implicated in various functions. Recently, hnRNP proteins have been shown to hinder communication between factors bound to different splice sites. hnRNP-U, also termed scaffold attachment factor A (SAF-A), binds to pre-mRNA and nuclear matrix/scaffold attachment region DNA elements.
The calculated molecular weight is 90 kDa However, the protein is usually observed at ~120 kDa (Dreyfuss, G., et al. (2002). Nat Rev Mol Cell Biol. 3(3):195-205.)
Immunogen
Epitope: Unknown
Recombinant protein corresponding to human hnRNP U.
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-hnRNP U antibody (Cat. # CS207320). ten percent of the precipitated proteins (lane 1: normal mouse IgG, lane 2: hnRNP U) and HeLa whole cell lysate (lane 3) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-hnRNP U antibody (Cat. # CS207320, 1:1000). Proteins were visualized using One-Step
Arrow indicates hnRNP U. (Figure 2).
Automated Microfluidics-based Electrophoretic RNA Separation and Analysis (MFE):
Representative lot data.
RIP Lysate prepared from HeLa cells (2 X 10E7 cell equivalents per IP) were subjected to immunoprecipitation using 5 µg of either 1. normal mouse IgG (Cat. # CS200621), or 2. Anti-hnRNP U antibody (Cat. # CS207320) and the Magna RIP
Successful immunoprecipitation of hnRNP U-associated RNA was verified by automated microfluidics-based electrophoretic RNA separation and analysis. Please refer to the Magna RIP
Western Blot Analysis:
Representative lot data.
K562 cell lysate was probed with Anti-hnRNP U, clone 3G6 (0.01 µg/mL). Proteins were visualized using a Goat Anti-Mouse IgG secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates hnRNP U (~120 kDa). (Figure 4).
Research Category
Epigenetics & Nuclear Function
Research Sub Category
RNA Metabolism & Binding Proteins
Apoptosis - Additional
This RIPAb+ hnRNP U -RIP Validated Antibody & Primer Set conveniently includes the hnRNP U antibody & the specific control PCR primers.
Biochem/physiol Actions
This antibody recognizes hnRNP U.
Packaging
10 assays per set. Recommended use: ~5 μg of antibody per RIP (dependent upon biological context).
Physical form
Anti-hnRNP U (Mouse Monoclonal), Part # CS207320. One vial containing 50 µg of protein G purified mouse IgG1 in 0.1M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide and 30% glycerol. Store at -20°C.
Normal Mouse IgG, Part # CS200621. 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 Primers, Ribosomal Protein S19, Part # CS207321. One vial containing 75 μL of 5 μM of each primer specific for human c-myc 3′ UTR. Store at -20°C.
FOR: ACG CGA GCT GCT TCC ACA G
REV: AGC TGC CAC CTG TCC GGC
Format: Purified
Protein G Purified
Preparation Note
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 Ribosomal Protein S19.
RNA Binding Protein Immunoprecipitation:
Representative lot data.
RIP Lysate prepared from HeLa 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-hnRNP U antibody and the Magna RIP® RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700).
Successful immunoprecipitation of hnRNP U-associated RNA was verified by qPCR using RIP Primers Ribosomal Protein S19, (Figure 1).
Please refer to the Magna RIP
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.
| biological source | mouse |
| Quality Segment | 100 |
| antibody form | purified immunoglobulin |
| clone | 3G6, monoclonal |
| species reactivity | human |
| manufacturer/tradename | RIPAb+,Upstate® |
| technique(s) | RIP: suitable,western blot: suitable |
| isotype | IgG2bκ |
| NCBI accession no. | NP_004492.2 |
| UniProt accession no. | Q00839 |
| shipped in | dry ice |
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