RIPAb+ p54nrb/NonO - RIP Validated Antibody and Primer Set

54 kDa

KLH-conjugated, synthetic peptide corres-ponding to amino acids TVREAGPPAFYRPNS

Immunoprecipitation from RIP lysate:
Representative lot data.
RIP lysate from HeLa cells (2 X 10⁶ cell equivalents per IP) was subjected to immunoprecipitation using either 0.5 µg of a normal mouse IgG or 0.5 µg of Anti-p54nrb/NONO antibody. Precipitated proteins (lane 1: rabbit IgG, lane 2: anti-p54nrb/NONO) and HeLa whole cell lysate (lane 3) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-p54nrb/NONO antibody (0.5 µg/mL).
Proteins were visualized using One-Step IP-Western kit (GenScript Cat. # L00231) (Please see figures).

Western Blot Analysis: 0.5-2.0 μg/mL of a previous lot detected p54nrb/NonO in RIPA lysates from MCF-7 cells (Please see figures).

Immunohistochemistry: This antibody has been reported by an independent laboratory to detect p54nrb/NonO in human breast tumor sections.

Immunoprecipitation: Reported by an independent lab to immunoprecipitate p54nrb/NonO.

Recognizes p54nrb/NonO, MW ~54 kDa.

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

Anti-p54nrb/NonO (Mouse Monoclonal IgG). One vial containing 50 µg of protein G purified mouse monoclonal IgG in 50 µL of buffer containing (0.1 M Tris-glycine, 0.15 M NaCl, pH 7.4, 0.05% sodium azide) and 30% glycerol. Store at -20°C.

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 Primers, FOS. One vial containing 75 μL of 5 μM of each primer specific for human FOS. 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

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.

RNA Binding Protein Immunoprecipitation:
RIP Lysate prepared from HeLa cells (2 X 10⁷ cell equivalents per IP) were subjected to immunoprecipitation using either 5 µg of a normal mouse IgG or 5 µg of Anti-p54nrb/NONO antibody and the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700).
Successful immunoprecipitation of p54nrb/NONO-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.

Function: DNA- and RNA binding protein, involved in several nuclear processes. Binds the conventional octamer sequence in double stranded DNA. Also binds single-stranded DNA and RNA at a site independent of the duplex site. Involved in pre-mRNA splicing, probably as an heterodimer with SFPQ. Interacts with U5 snRNA, probably by binding to a purine-rich sequence located on the 3′ side of U5 snRNA stem 1b. The SFPQ-NONO heteromer associated with MATR3 may play a role in nuclear retention of defective RNAs. The SFPQ-NONO heteromer may be involved in DNA unwinding by modulating the function of topoisomerase I/TOP1. The SFPQ-NONO heteromer may be involved in DNA nonhomologous end joining (NHEJ) required for double-strand break repair and V(D)J recombination and may stabilize paired DNA ends. In vitro, the complex strongly stimulates DNA end joining, binds directly to the DNA substrates and cooperates with the Ku70/G22P1-Ku80/XRCC5 (Ku) dimer to establish a functional preligation complex. Nono is involved in transcriptional regulation. The SFPQ-NONO-NR5A1/SF-1 complex binds to the CYP17 promoter and regulates basal and cAMP-dependent transcriptional avtivity. NONO binds to an enhancer element in long terminal repeats of endogenous intracisternal A particles (IAPs) and activates transcription.
Subunit structure: Monomer and component of the SFPQ-NONO complex, which is probably a heterotetramer of two 52 kDa (NONO) and two 100 kDa (SFPQ) subunits. NONO is a component of spliceosome and U5.4/6 snRNP complexes. Interacts with PSPC1 and SNRPA/U1A. Part of complex consisting of SFPQ, NONO and MATR3. Part of a complex consisting of SFPQ, NONO and NR5A1/SF-1. Part of a complex consisting of SFPQ, NONO and TOP1. Interacts with SPI1 By similarity. Interacts with RNF43.
Subcellular location: Nucleus.
Tissue specificity: Heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Also found in a number of breast tumor cell lines.
Post-translational modification: The N-terminus is blocked.
Involvement in disease: A chromosomal aberration involving NONO may be a cause of papillary renal cell carcinoma (PRCC). Translocation t(X;X)(p11.2;q13.1) with TFE3.
Sequence similarities: Contains 2 RRM (RNA recognition motif) domains.

Includes negative control mouse IgG antibody and control primers specific for human FOS.

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