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03-196 Sigma-AldrichRIPAb+™ EED - RIP Validated Antibody and Primer Set

This RIPAb+ EED -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

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References

Key Applications: RNA Binding Protein Immunoprecipitation (RIP), Western Blotting, Immunoprecipitation Research Category: Epigenetics & Nuclear Function, Apoptosis & Cancer Research Sub-Category: Chromatin Biology

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Key Spec Table

Species Reactivity	Key Applications
H, M	RIP, WB, IP

Description
Catalogue Number	03-196
Brand Family	Upstate
Trade Name	RIPAb+ Upstate
Description	RIPAb+™ EED - 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. Transcriptional repression mediated by EED involves histone deacetylation. EED protein is present in four isoforms. These EED isoforms selectively associate with distinct EZH2-containing complexes, resulting in differential targeting of their associated methyltransferase activity.
Alternate Names	WD protein associating with integrin cytoplasmic tails 1 embryonic ectoderm development
Background Information	Polycomb group proteins are important for maintaining transcriptional silencing. One conserved PcG complex, PRC2, is composed of several proteins including the histone methyltransferase EZH2, the WD-repeat protein EED (Embryonic ectoderm development), and the Zn-finger protein Suz12. Transcriptional repression mediated by EED involves histone deacetylation, while the EZH2 methylates histone H3 on lysine 27. EED protein is present in four isoforms. These EED isoforms selectively associate with distinct EZH2-containing complexes, resulting in differential targeting of their associated methyltransferase activity. These complexes play a role in Hox gene silencing, X-inactivation, germline development, stem cell pluripotency and cancer metastasis.

References

Product Information
Format	Purified
Control	Includes negative control normal mouse IgG antibody and control primers specific for the cDNA of human lincRNA SFPQ.
Presentation	Anti-EED (Mouse Monoclonal). One vial containing 50 μg of protein G purified antibody in 65 μL of 0.1 M Tris-Glycine (pH7.4) 150 mM NaCl, containing 0.05% 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, lincSFPQ. One vial containing 75 μL of 5 μM of each primer specific for the human lincRNA SFPQ. Store at -20°C. FOR: TCG TAC TGT TAG GCC CTT GG REV: AAC CTT GCA TGA AGA GCA CC
Quality Level	MQ100

Applications
Application	This RIPAb+ EED -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Key Applications	RNA Binding Protein Immunoprecipitation (RIP) Western Blotting Immunoprecipitation
Application Notes	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-EED antibody (Cat. # CS204372). Ten percent of the precipitated proteins (lane 1: mouse IgG, lane 3: EED) and HeLa whole cell lysate (lane 2) were resolved by electrophoresis, transferred to nitrocellulose and probed with Anti-EED antibody (Cat. # CS204372, 1:5000). Proteins were visualized using One-Step™ IP-Western kit (GenScript Cat. # L00232). Arrow indicates EED. Western Blot Analysis: Representative lot data. A431 cell lysate was resolved by electrophoresis, transferred to nitrocellulose and probed with Anti-EED (1:5,000 dilution). Proteins were visualized using a goat anti-mouse IgG conjugated to HRP and a chemiluminescence detection system. Arrow indicates EED (~50 kDa).

Biological Information
Immunogen	Recombinant fusion protein of full length murine EED tagged with MBP.
Concentration	Please refer to the Certificate of Analysis for the lot-specific concentration.
Host	Mouse
Specificity	This antibody recognizes mouse EED, (~30-65 kDa).
Isotype	IgG2aκ
Species Reactivity	Human Mouse
Species Reactivity Note	Human, Mouse. Not tested in other species.
Antibody Type	Monoclonal Antibody
Entrez Gene Number	NP_068676
Gene Symbol	HEED WAIT-1 WAIT1 hEED
Purification Method	Protein G Purified
UniProt Number	O75530
UniProt Summary	FUNCTION: Polycomb group (PcG) protein. Component of the PRC2/EED-EZH2 complex, which methylates 'Lys-9' and 'Lys-27' of histone H3, leading to transcriptional repression of the affected target gene. The PRC2/EED-EZH2 complex may also serve as a recruiting platform for DNA methyltransferases, thereby linking two epigenetic repression systems. Genes repressed by the PRC2/EED-EZH2 complex include HOXC8, HOXA9, MYT1 and CDKN2A. Ref.2 Ref.9 Ref.14 Ref.16 Ref.17 Ref.19 Ref.22 Ref.26 SUBUNIT: Interacts with MLL By similarity. Component of the PRC2/EED-EZH2 complex, which includes EED, EZH2, SUZ12, RBBP4 and RBBP7 and possibly AEBP2. The minimum components required for methyltransferase activity of the PRC2/EED-EZH2 complex are EED, EZH2 and SUZ12. The PRC2 complex may also interact with DNMT1, DNMT3A, DNMT3B and PHF1 via the EZH2 subunit and with SIRT1 via the SUZ12 subunit. Interacts with HDAC, HDAC2, histone H1 and YY1. May interact with ITGA4, ITGAE and ITGB7. May interact with the MA protein of HIV-1. SUBCELLULAR LOCATION: Nucleus. Note= Transiently colocalizes with XIST at inactive X chromosomes. Ref.2 Ref.17 Ref.3 Ref.12 Ref.15 SPECIFICITY: Expressed in brain, colon, heart, kidney, liver, lung, muscle, ovary, peripheral blood leukocytes, pancreas, placenta, prostate, spleen, small intestine, testis, thymus and uterus. Appears to be overexpressed in breast and colon cancer. Ref.2 Ref.3 Ref.1 Ref.8 Ref.20 Developmental stage Expression peaks at the G1/S phase boundary. Ref.14 Induction Expression is induced by E2F1, E2F2 and E2F3. Ref.14 SIMILARITY: Belongs to the WD repeat ESC family. Contains 7 WD repeats. Caution Two variants of the PRC2 complex have been described, termed PRC3 and PRC4. Each of the three complexes may include a different complement of EED isoforms, although the precise sequences of the isoforms in each complex have not been determined. The PRC2 and PRC4 complexes may also methylate 'Lys-26' of histone H1 in addition to 'Lys-27' of histone H3 (Ref.18 and Ref.20), although other studies have demonstrated no methylation of 'Lys-26' of histone H1 by PRC2 (Ref.21).
Molecular Weight	Ranges from 30-65 kDa (5-6 isoforms of EED are detected).

Physicochemical Information

Dimensions

Materials Information

Toxicological Information

Safety Information according to GHS

Safety Information

Product Usage Statements
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 mouse IgG or 5 µg of Anti-EED antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of EED-associated RNA was verified by qPCR using RIP Primers lincSFPQ. 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.

Product Usage Statements

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 mouse IgG or 5 µg of Anti-EED antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700).
Successful immunoprecipitation of EED-associated RNA was verified by qPCR using RIP Primers lincSFPQ.
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: Variabillity in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.

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: Variabillity 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
Catalogue Number	GTIN
03-196	04053252376498

Documentation

RIPAb+™ EED - RIP Validated Antibody and Primer Set SDS

Title
Safety Data Sheet (SDS)

RIPAb+™ EED - RIP Validated Antibody and Primer Set Certificates of Analysis

Title	Lot Number
RIPAb+ EED - 1977627	1977627
RIPAb+ EED - NRG1860701	NRG1860701
RIPAb+ EED - 2866771	2866771
RIPAb+ EED - 3178879	3178879
RIPAb+ EED - 3728377	3728377
RIPAb+ EED -2625877	2625877
RIPAb+ EED -2719914	2719914
RIPAb+ EED -2726757	2726757
RIPAb+ EED -2763861	2763861
RIPAb+ EED -2771409	2771409

References

Reference overview	Pub Med ID
Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks. Holdt, LM; Hoffmann, S; Sass, K; Langenberger, D; Scholz, M; Krohn, K; Finstermeier, K; Stahringer, A; Wilfert, W; Beutner, F; Gielen, S; Schuler, G; Gäbel, G; Bergert, H; Bechmann, I; Stadler, PF; Thiery, J; Teupser, D PLoS genetics 9 e1003588 2013 Show Abstract The chromosome 9p21 (Chr9p21) locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA) antisense non-coding RNA in the INK4 locus (ANRIL). ANRIL expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which ANRIL regulates target-genes in trans, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, trans-regulation was dependent on Alu motifs, which marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts. ANRIL bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon ANRIL over-expression. The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and atherogenic cell functions. ANRIL-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of ANRIL at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.	23861667

Reference overview

Pub Med ID

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