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03-200
Sigma-AldrichRIPAb+ SMN - RIP Validated Antibody and Primer Set
This RIPAb+ SMN -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
More>>This RIPAb+ SMN -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers. Less<<
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RIPAb+ SMN - RIP Validated Antibody and Primer Set
Overview
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.
The survival of motor neurons (SMN) protein is essential for the biogenesis of small nuclear RNA (snRNA)-ribonucleoproteins (snRNPs), the major components of the pre-mRNA splicing machinery. Though it is ubiquitously expressed, SMN deficiency causes the motor neuron degenerative disease spinal muscular atrophy (SMA). SMN deficiency, similar to that which occurs in severe SMA, has unexpected cell type-specific effects on the repertoire of snRNAs and mRNAs. It alters the stoichiometry of snRNAs and causes widespread pre-mRNA splicing defects in numerous transcripts of diverse genes, preferentially those containing a large number of introns, in SMN-deficient mouse tissues. These findings reveal a key role for the SMN complex in RNA metabolism and in splicing regulation and indicate that SMA is a general splicing disease that is not restricted to motor neurons.
References
Product Information
Format
Purified
Control
Includes negative control mouse IgG antibody and control primers specific for the cDNA of human U1snRNP.
Presentation
Anti-SMN (Mouse Monoclonal). One vial containing 50 µg of protein G purified monoclonal IgG1ĸ in buffer containing 0.1 M Tris-glycine, 150 mM NaCl, pH 7.4, 0.05% sodium azide before the addition of 30% glycerol. 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 Primers, U1snRNA. One vial containing 75 μL of 5 μM of each primer specific for the cDNA of U1 snRNP. Store at -20°C. FOR: GGG AGA TAC CAT GAT CAC GAA GGT REV: CCA CAA ATT ATG CAG TCG AGT TTC CC
This RIPAb+ SMN -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Key Applications
RNA Binding Protein Immunoprecipitation (RIP)
Western Blotting
Immunocytochemistry
Immunoprecipitation
Application Notes
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 either 5 µg of a normal mouse IgG or 5 µg of Anti-SMN antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of SMN-associated RNA was verified by qPCR using primers specific for human collagen alpha type 1 mRNA (Please see figures). Please refer to the Magna RIP™ (Cat. # 17-700) or EZ-Magna RIP™ (Cat. # 17-701) protocol for experimental details.
Immunoprecipitation from RIP lysate: Representative lot data. RIP lysate from HeLa cells (~2 X 10E7 cell equivalents per IP) was subjected to immunoprecipitation using either 5 µg of a normal mouse IgG or 5 µg of Anti-SMN antibody. Ten percent of the precipitated proteins (lane 2: mouse IgG, lane 4: SMN) and HeLa lysate (lane 3) were resolved by electrophoresis, transferred to PVDF and probed with anti-SMN antibody (1:1000 dilution). Proteins were visualized using One-Step™ IP-Western kit (GenScript®). Arrow indicates SMN (Please see figures).
Western Blot Analysis: Representative lot data. HeLa cell lysate was resolved by electrophoresis, transferred to PVDF membranes and probed with anti-SMN at a 0.5 ug/mL dilution. Proteins were visualized using a goat anti-mouse IgG conjugated to HRP using a chemiluminescence detection system. Arrow indicates SMN (~35 kDa) (Please see figures).
Immunocytochemistry Analysis: Representative lot data. A 1:500 dilution from a representative lot detected SMN in HeLa cells (Please see figures).
Biological Information
Immunogen
Histidine-tagged recombinant protein corresponding to human SMN.
Epitope
Unknown
Host
Mouse
Isotype
IgG1κ
Species Reactivity
Human
Mouse
Xenopus
Species Reactivity Note
Demonstrated to react with human. Predicted to react with mouse and xenopus based on sequence homology.
This gene is part of a 500 kb inverted duplication on chromosome 5q13. This duplicated region contains at least four genes and repetitive elements which make it prone to rearrangements and deletions. The repetitiveness and complexity of the sequence have also caused difficulty in determining the organization of this genomic region. The telomeric and centromeric copies of this gene are nearly identical and encode the same protein. However, mutations in this gene, the telomeric copy, are associated with spinal muscular atrophy; mutations in the centromeric copy do not lead to disease. The centromeric copy may be a modifier of disease caused by mutation in the telomeric copy. The critical sequence difference between the two genes is a single nucleotide in exon 7, which is thought to be an exon splice enhancer. Note that the nine exons of both the telomeric and centromeric copies are designated historically as exon 1, 2a, 2b, and 3-8. It is thought that gene conversion events may involve the two genes, leading to varying copy numbers of each gene. The protein encoded by this gene localizes to both the cytoplasm and the nucleus. Within the nucleus, the protein localizes to subnuclear bodies called gems which are found near coiled bodies containing high concentrations of small ribonucleoproteins (snRNPs). This protein forms heteromeric complexes with proteins such as SIP1 and GEMIN4, and also interacts with several proteins known to be involved in the biogenesis of snRNPs, such as hnRNP U protein and the small nucleolar RNA binding protein. Two transcript variants encoding distinct isoforms have been described. [provided by RefSeq]
FUNCTION: The SMN complex plays an essential role in spliceosomal snRNP assembly in the cytoplasm and is required for pre-mRNA splicing in the nucleus. It may also play a role in the metabolism of snoRNPs. SUBUNIT STRUCTURE: Component of an import snRNP complex composed of KPNB1, RNUT1, SMN1 and ZNF259. Part of the core SMN complex that contains SMN1, SIP1/GEMIN2, DDX20/GEMIN3, GEMIN4, GEMIN5, GEMIN6, GEMIN7, GEMIN8 and STRAP/UNRIP. Interacts with DDX20, FBL, NOLA1, RNUT1, SYNCRIP and with several spliceosomal snRNP core Sm proteins, including SNRPB, SNRPD1, SNRPD2, SNRPD3, SNRPE and ILF3. Interacts with OSTF1. SUBCELLULAR LOCATION: Cytoplasm. Nucleus › gem. Note: Localized in subnuclear structures next to coiled bodies, called Gemini of Cajal bodies (Gems). TISSUE SPECIFICITY: Expressed in a wide variety of tissues. Expressed at high levels in brain, kidney and liver, moderate levels in skeletal and cardiac muscle, and low levels in fibroblasts and lymphocytes. Also seen at high levels in spinal cord. Present in osteoclasts and mononuclear cells (at protein level). INVOLVEMENT IN DISEASE: Defects in SMN1 are the cause of spinal muscular atrophy autosomal recessive type 1 (SMA1) [MIM:253300]. Spinal muscular atrophy refers to a group of neuromuscular disorders characterized by degeneration of the anterior horn cells of the spinal cord, leading to symmetrical muscle weakness and atrophy. Autosomal recessive forms are classified according to the age of onset, the maximum muscular activity achieved, and survivorship. The severity of the disease is mainly determined by the copy number of SMN2, a copy gene which predominantly produces exon 7-skipped transcripts and only low amount of full-length transcripts that encode for a protein identical to SMN1. Only about 4% of SMA patients bear one SMN1 copy with an intragenic mutation. SMA1 is a severe form, with onset before 6 months of age. SMA1 patients never achieve the ability to sit. Defects in SMN1 are the cause of spinal muscular atrophy autosomal recessive type 2 (SMA2) [MIM:253550]. SMA2 is an autosomal recessive spinal muscular atrophy of intermediate severity, with onset between 6 and 18 months. Patients do not reach the motor milestone of standing, and survive into adulthood. Defects in SMN1 are the cause of spinal muscular atrophy autosomal recessive type 3 (SMA3) [MIM:253400]. SMA3 is an autosomal recessive spinal muscular atrophy with onset after 18 months. SMA3 patients develop ability to stand and walk and survive into adulthood. Defects in SMN1 are the cause of spinal muscular atrophy autosomal recessive type 4 (SMA4) [MIM:271150]. SMA4 is an autosomal recessive spinal muscular atrophy characterized by symmetric proximal muscle weakness with onset in adulthood and slow disease progression. SMA4 patients can stand and walk. MISCELLANEOUS: The SMN gene is present in two highly homologous and functional copies (TelSMN/SMN1 and CenSMN/SMN2). The telomeric copy of SMN gene (TelSMN/SMN1) seems to be the SMA-determining gene while the centromeric copy seems unaffected. SEQUENCE SIMILARILITIES: Belongs to the SMN family. Contains 1 Tudor domain.
Molecular Weight
~35 kDa was observed; however, the calculated molecular weight is 31.849 kDa.
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Quality Assurance
RNA Binding Protein Immunoprecipitation: RIP Lysate prepared from HeLa cells (~2 X 10E7 cell equivalents per IP) were subjected to immunoprecipitation using either 5 µg of a normal mouse IgG or 5 µg of Anti-SMN antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of SMN-associated RNA was verified by qPCR using RIP Primers U1snRNA (Please see figures). Please refer to the Magna RIP™ (Cat. # 17-700) or EZ-Magna RIP™ (Cat. # 17-701) protocol for experimental details.
Usage Statement
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.
Storage and Shipping Information
Storage Conditions
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.
Packaging Information
Material Size
10 assays
Material Package
10 assays per set. Recommended use: ~5 μg of antibody per RIP (dependent upon biological context).
Transport Information
Supplemental Information
Specifications
Global Trade Item Number
Bestellnummer
GTIN
03-200
04053252480621
Documentation
RIPAb+ SMN - RIP Validated Antibody and Primer Set Analysenzertifikate
