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For Research Use Only. RN028P Page 1 of 4 Not for use in diagnostic procedures. RIP-Certified Antibody Anti-EIF2C1/AGO1 Code No. Quantity Concentration Form RN028P 200 µµµL 1 mg/mL Affinity Purified BACKGROUND: Eukaryotic translation initiation REACTIVITY: This antibody reacts with human and factor 2C, subunit 1 (EIF2C1; AGO1) is a member of the mouse EIF2C1 on Western blotting, Immunoprecipitation argonaute protein family. It contains Piwi/Argonaute/Zwille and RNP Immunoprecipitation. (PAZ) and Piwi domain. EIF2C1 and EIF2C2 form the RNA-induced silencing complex that mediates gene APPLICATIONS: silencing by RNA interference. EIF2C1 is also known as RNP Immunoprecipitation ; 15 µg/500 µL of cell extract the initiator of RNA-induced transcriptional gene silencing from 1.3 x 10 7 cells (TGS) in human cells. EIF2C1, DNA methyltransferase 3a, Western blotting ; 1:1,000 for chemiluminescence detection and histone deacetylase I form the transcriptional silencing system complex. EIF2C1 binds to RNA polymerase II and Immunoprecipitation ; 5 µg/500 µL of cell extract from mediates TGS through heterochromatin formation. In 5 x 10 6 cells humans, the gene encoding EIF2C1 is localized on Immunohistochemistry ; Not tested chromosome 1; this region is often deleted in cases of Immunocytochemistry ; Not tested Wilms’ tumors. EIF2C1 may be involved in tumor Flow cytometry ; Not tested development because its expression is upregulated in tumors that lack the Wilm’s tumor suppressor gene, namely, Detailed procedures are provided in the following WT1. PROTOCOLS . RIP-CERTIFIED ANTIBODY: INTENDED USE: Posttranscriptional regulation of gene expression is a For Research Use Only. Not for use in diagnostic procedures. ribonucleoprotein-driven process, which involves RNA binding proteins (RBPs) and non-coding RNAs that affect splicing, nuclear export, subcellular localization, mRNA REFERENCES: decay and translation. The RNP Immunoprecipitation-Chip 1) Kim, D. H., et al., Nat. Struct. Mol. Biol. 13, 793-797 (2006) (RIP-Chip), RIP-Seq and RIP-RTPCR allow the 2) Hawkins, P. G., et al., Nucleic Acids Res. 37, 2984-2995 (2009) identification of multiple RNA targets of RBPs globally 3) Martinez, J., et al., Cell 110, 563-574 (2002) and within the context of a cell extract. Antibodies specific 4) Carmell, M. A., et al., Genes Dev. 16, 2733-2742 (2002) to the RNA binding protein of interest are used to co-immunoprecipitate the RNA binding protein and the SPECIES CROSS REACTIVITY: associated subset of RNAs. The RNA content is interrogated using standard microarray or sequencing Species Human Mouse Rat Hamster technology. RIP-Certified Antibody is validated for use in 293T, HeLa Cell NIH/3T3 Rat1 Not Tested RNP Immunoprecipitation (RIP) in conjunction with the K562, Jurkat RIP-Assay Kit for microRNA distributed from MBL. Its ability to immunoprecipitate RNAs and RBPs complex was Reactivity on IP + + − confirmed by quantitative and qualitative analysis on NanoDrop, Bioanalyzer and RT-PCR or microarray. LICENSING OPPORTUNITY: The RIP-Assay uses SOURCE: This antibody was purified from rabbit serum patented technology (US patent No. 6,635,422, US patent by affinity column chromatography. The rabbit was No. 7,504,210) of Ribonomics, Inc. MBL manufactures and immunized with KLH conjugated synthetic peptide, distributes this product under license from Ribonomics, Inc. corresponding to N-terminus of human EIF2C1. Researchers may use this product for the purposes of their own research. Researchers are not allowed to use this product or RIP-Assay technology for commercial purpose FORMULATION: 200 µL volume of PBS containing without acquiring a license. For commercial use and 50% glycerol, pH 7.2. No preservative is contained. licensing opportunities, please contact us at [email protected] STORAGE: This antibody solution is stable for one year o from the date of purchase when stored at -20 C. MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL https://ruo.mbl.co.jp/je/rip-assay/ e-mail [email protected], TEL 052-238-1904 RN028P Page 2 of 4 7) Add 500 µL of cell lysate (precleared sample of step 4), nt 1 2 o then incubate with gentle agitation for 3 hours at 4 C. 100 8) Wash the beads 4 times with mi-Wash Buffer (+) 75 (centrifuge the tube at 2,000 x g for 1 minute). 9) Add 250 µL of Master mix solution (mi-Solution I: 50 mi-Solution II = 10 µL: 240 µL). Vortex thoroughly, then spin-down. 40 10) Add 150 µL of mi-Solution III. Vortex thoroughly. 30 11) Centrifuge the tube at 2,000 x g for 2 minutes. 12) Transfer the supernatant to the fresh tube containing 2 miRNA 20 µL of mi-Solution IV. 13) Add 300 µL of ice-cold 2-propanol, vortex for 10 seconds. Place at -20 oC for 20 minutes. Centrifuge the tube at 12,000 x g for 10 minutes. Analysis of isolated small RNA (including miRNA) from Jurkat by silver staining 14) Transfer the supernatant, which contains small RNAs, to following denaturing PAGE. the tube containing 2 µL of mi-Solution IV. Isolation Lane1: Normal Rabbit IgG method for small RNAs from the supernatant is Lane2: RN028P described in the following step 15. In case of purification for large RNAs in the pellet, skip to step 16. 15) Add 500 µL of ice-cold 2-propanol, vortex for 10 PROTOCOLS: seconds, then place at -20 oC for 20 minutes. Centrifuge RNP Immunoprecipitation the tube at 12,000 x g for 10 minutes. Some buffers and reagents are included in the RIP-Assay Kit 16) Wash the pellet 2 times with 0.5 mL of ice-cold 70% for microRNA (code. RN1005). Please also refer to the ethanol and dry up the pellet for 5-15 minutes. protocol packaged in the RIP-Assay Kit for microRNA . 17) Dissolve the pellets in nuclease-free water. [Material Preparation] (Positive control for RNP Immunoprecipitation; Jurkat) 1. mi-Lysis Buffer (+) Before using the mi-Lysis Buffer, protease inhibitors, 1 2 3 4 5 RNase inhibitors, and DTT are added to the mi-Lysis kDa Buffer at the appropriate concentration. 2. mi-Wash Buffer (+) 150 Before using the mi-Wash Buffer, DTT is added to the mi-Wash Buffer at the appropriate concentration. 100 EIF2C1 Protocol (RNA isolation: Separation method) 1) Wash 1.3 x 10 7 cells 4 times with PBS and resuspend 75 them with 500 µL of ice-cold mi-Lysis Buffer (+) containing appropriate protease inhibitors, RNase inhibitors, and DTT. Vortex thoroughly, then incubate it Western blot analysis of EIF2C1 on ice for 10 minutes. expression in 293T (1), HeLa (2), K562 2) Centrifuge the tube at 12,000 x g for 5 minutes at 4 oC (3), Jurkat (4) and NIH/3T3 (5) using and transfer the supernatant to another tube. RN028P. 3) Add 30 µL of 50% protein A agarose beads slurry resuspended in mi-Lysis Buffer (+) into the supernatant. Incubate it at 4 oC with rotating for 1 hour. SDS-PAGE & Western Blotting 7 4) Centrifuge the tube at 2,000 x g for 1 minute at 4 oC and 1) Wash cells (approximately 1 x 10 cells) 3 times with transfer the supernatant to another fresh tube (precleared PBS and suspend them in 1 mL of Laemmli’s sample sample). buffer. 5) Mix 30 µL of 50% protein A agarose beads slurry 2) Boil the samples for 2 minutes and centrifuge. Load 10 resuspended in nuclease-free PBS with Normal Rabbit µL of sample per lane on a 1-mm-thick IgG (RIP-Assay Kit for microRNA ) or SDS-polyacrylamide gel and carry out electrophoresis. anti-EIF2C1/AGO1 polyclonal antibody at the 3) Blot the protein to a polyvinylidene difluoride (PVDF) 2 concentration suggested in the APPLICATIONS, and membrane at 1 mA/cm for 1 hour in a semi-dry transfer then add 1 mL of mi-Wash Buffer (+) into each tube. system (Transfer Buffer: 25 mM Tris, 190 mM glycine, Incubate with gently agitation for 1 hour at 4 oC. 20% MeOH). See the manufacturer's manual for precise 6) Wash the beads once with ice-cold mi-Lysis Buffer (+) transfer procedure. (centrifuge the tube at 2,000 x g for 1 minute). Carefully 4) To reduce nonspecific binding, soak the membrane in discard the supernatant using a pipettor without 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room o disturbing the beads. temperature, or overnight at 4 C. RN028P Page 3 of 4 5) Incubate the membrane with primary antibody diluted 6) Wash the beads once with ice-cold Lysis Buffer with PBS, pH 7.2 containing 1% skimmed milk as (centrifuge the tube at 2,000 x g for 1 minute). Carefully suggested in the APPLICATIONS for 1 hour at room discard the supernatant using a pipettor without temperature. (The concentration of antibody will depend disturbing the beads. on the conditions.) 7) Add 500 µL of cell lysate (precleared sample of step 4), 6) Wash the membrane with PBS-T [0.05% Tween-20 in then incubate with gentle agitation for 3 hours at 4 oC PBS] (5 minutes x 3 times). 8) Wash the beads 4 times with Lysis Buffer (centrifuge the 7) Incubate the membrane with the 1:10,000 tube at 2,000 x g for 1 minute). HRP-conjugated anti-rabbit IgG (MBL; code no. 458) 9) Resuspend the beads in 20 µL of Laemmli’s sample diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. hour at room temperature. Use 20 µL/lane for the SDS-PAGE analysis. 8) Wash the membrane with PBS-T (5 minutes x 3 times). (See SDS-PAGE & Western blotting .) 9) Wipe excess buffer off the membrane, and incubate membrane with an appropriate chemiluminescence (Positive control for Immunoprecipitation; 293T) reagent for 1 minute.
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    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 14 July 2011 (14.07.2011) WO 2011/085347 A2 (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, C12N 15/113 (2010.01) A61K 48/00 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, A61K 31/7088 (2006.01) C12N 15/63 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (21) International Application Number: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, PCT/US201 1/020768 ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (22) International Filing Date: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 11 January 201 1 ( 11.01 .201 1) SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, 61/293,739 11 January 2010 ( 11.01 .2010) US ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (71) Applicant (for all designated States except US): OPKO EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, CURNA, LLC [US/US]; 440 Biscayne Boulevard, Mia LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, mi, FL 33 137 (US).
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    The Argonaute Family: Tentacles That Reach Into Rnai, Developmental Control, Stem Cell Maintenance, and Tumorigenesis

    Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis Michelle A. Carmell,1,2,3 Zhenyu Xuan,1,3 Michael Q. Zhang,1 and Gregory J. Hannon1,4 1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA; 2Program in Genetics, State University of New York at Stony Brook, Stony Brook, New York 11794, USA RNA interference (RNAi) is an evolutionarily conserved The Argonaute family process through which double-stranded RNA (dsRNA) Argonaute proteins make up a highly conserved family induces the silencing of cognate genes (for review, see whose members have been implicated in RNAi and re- Bernstein et al. 2001b; Carthew 2001). Sources of dsRNA lated phenomena in several organisms. In addition to silencing triggers include experimentally introduced roles in RNAi-like mechanisms, Argonaute proteins in- dsRNAs, RNA viruses, transposons, and RNAs tran- fluence development, and at least a subset are involved scribed from complex transgene arrays (for review, see in stem cell fate determination. Argonaute proteins are Hammond et al. 2001b). Short hairpin sequences en- ∼100-kD highly basic proteins that contain two common coded in the genome also appear to enter the RNAi path- domains, namely PAZ and PIWI domains (Cerutti et al. way and function to regulate the expression of endog- 2000). The PAZ domain, consisting of 130 amino acids, enous, protein-coding genes (Grishok et al. 2001; has been identified in Argonaute proteins and in Dicer Hutvagner et al. 2001; Ketting et al.