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Key Specifications Table
| Species Reactivity | Key Applications |
|---|---|
| H, M, Chp, Rhesus Macaque, Ca | ICC, IH(P), WB, RIP |
| Description | |
|---|---|
| Catalogue Number | 03-241 |
| Trade Name |
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| Description | RIPAb+™ PUM2 Antibody |
| 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. PUM2 is a sequence-specific RNA-binding protein that regulates translation and mRNA stability by binding to the 3-UTR of mRNA targets. The PUM class of proteins plays an active role in the sustainability of mitotic stem cell proliferation and the regulation of cell development and differentiation. |
| Alternate Names |
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| Background Information | PUM2 is a member of the Puf family of RNA-binding proteins, which are believed to play a role in the regulation of posttranscriptional gene expression. Members of the Puf family of RNA binding proteins function as repressors of translation when bound to the 3-untranslated region of RNA transcripts. |
| Product Information | |
|---|---|
| Format | Affinity Purified |
| Control |
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| Presentation | Components: Anti-PUM2 (Rabbit Polyclonal). One vial containing 50 µg of affinity purified polyclonal in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl, with 0.05% sodium azide, before the addition of 30% glycerol. Store at -20°C. Concentration: 0.7 mg/mL Normal Rabbit IgG. One vial containing 125 µg Rabbit IgG in 125 µL storage buffer containing 0.05% sodium azide. Store at -20°C. RIP Primers, NFKBIA mRNA. One vial containing 75 μL of each primer (5 μM) specific for a region of the human NFKBIA mRNA. Store at -20°C. FOR: GCT ATT CTC CCT ACC AGC TC REV: CAG TCA TCA TAG GGC AGC TC |
| Quality Level | MQ100 |
| Applications | |
|---|---|
| Application | Detect RIPAb+ PUM2 using this RIPAb+ PUM2 Antibody validated for use in ICC, IHC(P), Western Blotting, RNA Binding Protein Immunoprecipitation (RIP). |
| Key Applications |
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| Application Notes | Western Blot Analysis: Representative lot data. Mouse embryonic stem cell lysate was resolved by electrophoresis, transferred to PVDF membranes and probed with Anti-Pumilio 2 (PUM2) 1 µg/mL. Proteins were visualized using a Donkey Anti-Rabbit IgG conjugated to HRP and chemiluminescence detection system. Arrow indicates PUM2 (~120 kDa). Note: A non-specific band may be observed at ~48 kDa in some lysates. (Figure 2). Immunohistochemistry (Parafin) Analysis: Representative lot data. Paraffin-embedded human testis (Fig. 3A) and human teratoma (Fig. 3B) tissues were prepared using heat-induced epitope retrieval in citrate buffer, pH 6.0. Immunostaining was performed using a 1:400 dilution of Anti-Pumilio 2 (PUM2). Reactivity was detected using the IHC-Select Detection Kit (Cat. No. DAB050). Staining pattern appears to be restricted to primary spermatocytes with no Sertoli cell involvement, as expected. Immunocytochemistry Analysis: Representative lot data. Confocal fluorescent analysis of mouse and human embryonic stem cells using Anti-Pumilio 2 (PUM2) (Red). Actin filaments have been labeled with Alexa Fluor® 488 dye - Phalloidin (Green). Nucleus is stained with DAPI (Blue). (Figure 4) This antibody positively stains the nucleus and cytosol. |
| Biological Information | |
|---|---|
| Immunogen | KLH-conjugated linear peptide corresponding to the N-terminus of human Pumilio-2. |
| Epitope | N-terminus |
| Concentration | Please refer to the Certificate of Analysis for the lot-specific concentration. |
| Host | Rabbit |
| Specificity | This antibody recognizes Pumilio-2 near the N-terminus. |
| Isotype | IgG2a |
| Species Reactivity |
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| Species Reactivity Note | Demonstrated to react with human and mouse. Predicted to react with chimpanzee, rhesus monkey, and canine based on 100% sequence homology. |
| Antibody Type | Polyclonal Antibody |
| Entrez Gene Number |
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| Gene Symbol |
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| Purification Method | Affinity Purfied |
| UniProt Number |
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| UniProt Summary | FUNCTION: Sequence-specific RNA-binding protein that regulates translation and mRNA stability by binding the 3-UTR of mRNA targets. Its interactions and tissue specificity suggest that it may be required to support proliferation and self-renewal of stem cells by regulating the translation of key transcripts. SUBUNIT STRUCTURE: Homodimer; homodimerizes in vitro. Interacts with DAZ, DAZL and NANOS1 via its pumilio repeats. Binds to a RNA consensus sequence, that is related to the Nanos Response Element (NRE), a 16 bp sequence found in the 3-UTR of the Drosophila hb mRNA. Also binds to the NRE. SUBCELLULAR LOCATION: Cytoplasm Probable. TISSUE SPECIFICITY: Highly expressed in testis and ovary. Predominantly expressed in stem cells and germ cells. Expressed at lower level in brain, heart, kidney, liver, muscle, placenta, intestine and stomach Expressed in cerebellum, corpus callosum, caudate nucleus, hippocampus, medulla oblongata and putamen. Expressed in all fetal tissues tested. INDUCTION: Down-regulated in keratinocytes upon UV B radiation. DOMAIN: The pumilio repeats mediate the association with RNA by packing together to form a right-handed superhelix that approximates a half donut. The number as well as the specific sequence of the repeats determine the specificity for target mRNAs By similarity. SEQUENCE SIMILARITIES: Contains 1 PUM-HD domain. Contains 8 pumilio repeats. |
| Molecular Weight | ~120 kDa observed. Non specific band may be observed at 48 kDa. |
| Product Usage Statements | |
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| Quality Assurance | 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 rabbit IgG or 5 µg of Anti-PUM2 antibody and the Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of PUM2-associated RNA was verified by qPCR using RIP Primers, NFKBIA mRNA, (Figure 1). Please refer to the Magna RIP™ (Cat. # 17-700) or EZ-Magna RIP™ (Cat. # 17-701) protocol for experimental details. |
| Usage Statement |
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| Storage and Shipping Information | |
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| Storage Conditions | Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon first thaw, 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). |