For Research Use Only. RN045P Not for use in diagnostic procedures. Page 1 of 4 RIP-Certified Antibody Anti-SLBP Code No. Quantity Concentration Form RN045P 200 L 1 mg/mL Affinity Purified BACKGROUND: The stem-loop binding protein APPLICATIONS: (SLBP) is an RNA binding protein known as a cell-cycle RNP Immunoprecipitation; 15 g/500 L of cell extract regulator. SLBP binds to the stem-loop structure of from 2.1 x 107 cells replication-dependent histone mRNA that has neither Western blotting; 1:1,000 for chemiluminescence detection introns nor poly(A) tail. SLBP is required for histone system pre-mRNA processing, histone mRNA export, translation, Immunoprecipitation; 1.5 g/500 L of cell extract from and degradation. SLBP is accumulated just before entry of 2.1 x 106 cells S phase and degraded at S/G2 transition. Cyclin A/Cdk1 Immunohistochemistry; Not tested modulates degradation of SLBP at the end of S phase, Immunocytochemistry; Not tested which leads to an inhibition of histone mRNA biosynthesis. Flow cytometry; Not tested RIP-CERTIFIED ANTIBODY: Detailed procedures are provided in the following Posttranscriptional regulation of gene expression is a PROTOCOLS. ribonucleoprotein-driven process, which involves RNA binding proteins (RBPs) and non-coding RNAs that affect REFERENCES: splicing, nuclear export, subcellular localization, mRNA 1) Sullivan, K. D., et al., RNA 15, 459-472 (2009) decay and translation. The RNP Immunoprecipitation-Chip 2) Koseoglu, M. M., et al., Mol. Cell Biol. 28, 4469-4479 (2008) (RIP-Chip), RIP-Seq and RIP-RTPCR allow the 3) Dominski, Z., et al., Genes Dev. 16, 58-71 (2002) identification of multiple RNA targets of RBPs globally 4) Wang, Z. F., et al., Genes Dev. 10, 3028-3040 (1996) and within the context of a cell extract. Antibodies specific to the RNA binding protein of interest are used to SPECIES CROSS REACTIVITY: co-immunoprecipitate the RNA binding protein and the associated subset of mRNAs. The mRNA content is Species Human Mouse Rat Hamster interrogated using standard microarray or sequencing 293T, HeLa, technology. RIP-Certified Antibody is validated for use in NIH/3T3, Cells K562, Jurkat, Rat1 CHO WR19L RNP Immunoprecipitation (RIP) in conjunction with the MCF7 RIP-Assay Kit distributed from MBL. Its ability to Reactivity on + + + + immunoprecipitate mRNAs and RBPs complex was WB confirmed by quantitative and qualitative analysis on NanoDrop, Bioanalyzer and RT-PCR or microarray. LICENSING OPPORTUNITY: The RIP-Assay uses patented technology (US patent No. 6,635,422, US patent SOURCE: This antibody was purified from rabbit serum No. 7,504,210) of Ribonomics, Inc. MBL manufactures and by affinity column chromatography. The rabbit was distributes this product under license from Ribonomics, Inc. immunized with KLH conjugated synthetic peptide, Researchers may use this product for their own research. corresponding to C-terminus of human SLBP. Researchers are not allowed to use this product or RIP-Assay technology for commercial purpose without a FORMULATION: 200 L volume of PBS containing license. For commercial use, please contact us for licensing 50% glycerol, pH 7.2. No preservative is contained. opportunities at [email protected] STORAGE: This antibody solution is stable for one year o from the date of purchase when stored at -20 C. REACTIVITY: This antibody reacts with human SLBP on Western blotting, Immunoprecipitation and RNP Immunoprecipitation. Other species cross reactivity is confirmed by Western blotting. INTENDED USE: For Research Use Only. Not for use in diagnostic procedures. MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL https://ruo.mbl.co.jp/je/rip-assay/ e-mail [email protected], TEL 052-238-1904 RN045P Page 2 of 4 Normal Rabbit IgG Anti-SLBP resuspended in nuclease-free PBS with Normal Rabbit IgG (RIP-Assay Kit) or anti-SLBP antibody at the concentration as suggested in the APPLICATIONS, and then add 1 mL of Wash buffer (+) into each tube. Incubate with gently agitation for 1 hour at 4oC. 6) Wash the beads once with ice-cold Lysis Buffer (+) RNA Intensity RNA (centrifuge the tube at 2,000 x g for 1 minute). Carefully Nucleotide length discard the supernatant using a pipettor without disturbing the beads. G 7) Add 500 L of cell lysate (precleared sample of step 4), Ig o then incubate with gentle agitation for 3 hours at 4 C. it b b 8) Wash the beads 4 times with Wash Buffer (+) (centrifuge a P R A B N the tube at 2,000 x g for 1 minute). r l L e a R -S l d m i a 9) Add 400 L of Master mix solution (Solution I: Solution d r t t a o n o L N a T II = 10 L: 390 L). Vortex thoroughly, then spin-down. 10) Add 250 L of Solution III. Vortex thoroughly. 28 S 11) Centrifuge the tube at 2,000 x g for 2 minutes. 18 S 12) Transfer the supernatant to the fresh tube containing 2 L of Solution IV. 13) Add 600 L of ice-cold 2-propanol to each tube, vortex briefly but thoroughly, then spin-down. Incubate the tube at -20oC for 20 minutes. Centrifuge the tube at 12,000 x g Analysis of isolated RNA with Bioanalyzer. for 10 minutes. Average of the RNA Quantity (n=2) 14) Wash the pellet 2 times with 0.5 mL of ice-cold 70% Antibody RNA (ng) Ethanol and dry up the pellet for 5-15 minutes. 15) Dissolve the pellets in nuclease-free water. Normal Rabbit IgG 76.5 16) RNA was quantified with NanoDrop (Thermo Fisher anti-SLBP 290.4 Scientific Inc.) and the RNA quality was analyzed with Total RNA 525585.0 Bioanalyzer (Agilent Technologies, Inc.). PROTOCOLS: (Positive control for RNP Immunoprecipitation; 293T) RNP Immunoprecipitation 123456789 Some buffers and reagents are included in the RIP-Assay Kit kDa (code. RN1001). Please also refer to the protocol packaged in the RIP-Assay Kit. 75 50 [Material Preparation] SLBP 1. Lysis Buffer (+) 37 Before using the Lysis Buffer, protease inhibitors, RNase inhibitors, and DTT are added to the Lysis Buffer at the appropriate concentration. 2. Wash Buffer (+) 25 Before using the Wash Buffer, DTT is added to the Wash Buffer at the appropriate concentration. Western blot analysis of SLBP expression in 293T (1), HeLa (2), K562 Protocol (3), Jurkat (4), MCF7 (5), NIH/3T3 (6), 1) Wash 2.1 x 107 cells 4 times with PBS and resuspend WR19L (7), Rat1 (8) and CHO (9) using them with 500 L of ice-cold Lysis Buffer (+) containing RN045P. appropriate protease inhibitors, RNase inhibitors, and DTT. Vortex thoroughly, then incubate it on ice for 10 SDS-PAGE & Western Blotting minutes. 1) Wash 1 x 107 cells 3 times with PBS and suspend them in 2) Centrifuge the tube at 12,000 x g for 5 minutes at 4oC and 1 mL of Laemmli’s sample buffer. transfer the supernatant to another tube. 2) Boil the samples for 2 minutes and centrifuge. Load 10 L 3) Add 25 L of 50% protein A agarose beads slurry of sample per lane on a 1-mm-thick SDS-polyacrylamide resuspended in Lysis Buffer (+) into the supernatant. gel and carry out electrophoresis. Incubate it at 4oC with rotating for 1 hour. 3) Blot the protein to a polyvinylidene difluoride (PVDF) 4) Centrifuge the tube at 2,000 x g for 1 minute at 4oC and membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer transfer the supernatant to another fresh tube (precleared system (Transfer Buffer: 25 mM Tris, 190 mM glycine, sample). 20% MeOH). See the manufacturer's manual for precise 5) Mix 25 L of 50% protein A agarose beads slurry transfer procedure. RN045P Page 3 of 4 4) To reduce nonspecific binding, soak the membrane in 5% transfer the supernatant to another tube (precleared skimmed milk (in PBS, pH 7.2) for 1 hour at room sample). temperature, or overnight at 4oC. 5) Mix 25 L of 50% protein A agarose beads slurry 5) Incubate the membrane with primary antibody diluted resuspended in PBS with Normal Rabbit IgG (RIP-Assay with PBS, pH 7.2 containing 1% skimmed milk as Kit) or anti-SLBP antibody at the concentration suggested suggested in the APPLICATIONS for 1 hour at room in the APPLICATIONS, and then add 1 mL of Wash temperature. (The concentration of antibody will depend buffer into each tube. Incubate with gently agitation for 1 on the conditions.) hour at 4oC. 6) Wash the membrane with PBS-T [0.05% Tween-20 in 6) Wash the beads once with ice-cold Lysis Buffer PBS] (5 minutes x 3 times). (centrifuge the tube at 2,000 x g for 1 minute). Carefully 7) Incubate the membrane with the 1:5,000 HRP-conjugated discard the supernatant using a pipettor without disturbing anti-rabbit IgG (MBL; code no. 458) diluted with 1% the beads. skimmed milk (in PBS, pH 7.2) for 1 hour at room 7) Add 500 L of cell lysate (precleared sample of step 4), temperature. then incubate with gentle agitation for 3 hours at 4oC 8) Wash the membrane with PBS-T (5 minutes x 3 times). 8) Wash the beads 4 times with Wash Buffer (centrifuge the 9) Wipe excess buffer off the membrane, and incubate tube at 2,000 x g for 1 minute). membrane with an appropriate chemiluminescence 9) Resuspend the beads in 20 L of Laemmli’s sample buffer, reagent for 1 minute. boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10 10) Remove extra reagent from the membrane by dabbing L/lane for the SDS-PAGE analysis. with a paper towel, and seal it in plastic wrap.