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Applikationshinweise
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RNP Immunoprecipitation RNP Immunoprecipitation, 15 ug/500 uL of cell extract 7 from 1 x 10 cells Western blotting, 1:1,000 for chemiluminescence detection system Immunoprecipitation, 5 ug/250 uL of cell extract from 2.5 x 10 6 cells Immunohistochemistry, Not tested Immunocytochemistry, Not tested Flow cytometry, Not tested . Protocol: RNP Immunoprecipitation Some buffer and reagents are included in the RIP-Assay Kit (code. RN1001). Please also refer to the protocol packaged the RIP-Assay Kit. n] 1. ded to the Lysis Buffer at the ntration. 2. dded to the Wash Buffer at the appropriate concentration. in [Material Preparatio Lysis Buffer (+) Before using the Lysis Buffer, protease inhibitors, RNase inhibitors, and DTT are adappropriate conceWash Buffer (+) Before using the Wash Buffer, DTT is a Protocol 1) Wash 1 x 10 7 cells 2 times with PBS and resuspend them with 500 uL of ice-cold Lysis Buffer (+) containing appropriate protease inhibitors, RNase inhibitors, and DTT. Vortex for 10 seconds. Leave on ice for 10 minutes. 2) Centrifuge the tube at 12,000 x g for 5 minutes at 4 o C and e supernatan the supernatant to another tube (precleare ion for 1 hour at 4 C. t. transfer the supernatant to another tube. 3) Add 25 uL of 50% protein A agarose beads slurry resuspended in Lysis Buffer (+) into th Incubate it at 4 o C with rotating for 1 hour. 4) Centrifuge the tube at 2,000 x g for 1 minute at 4 o C and transfer d sample). 5) Mix both 25 uL of 50% protein A agarose beads slurry resuspended in nuclease-free PBS and Normal Rabbit IgG (RIP-Assay Kit) or anti-STAU2 antibody at the amount of suggested in the APPLICATIONS, and then add 1 mL of Wash buffer (+) into each tube. Incubate with gently agitat o RN A I nten sity Nucleotide length Normal Rabbit IgG anti-STAU2 6) Wash the beads once with ice-cold Lysis Buffer (+) (centrifuge the tube at 2,000 x g for 1 minute). Carefully discard the supernatant using a pipettor without disturbing the beads. Analysis of isolated RNA with Bioanalyzer. La dd er an ti- S TA U 2 To ta l R N A 28 S 18 S N or m al R ab bi t I gG 7) Add 500 uL of cell lysate (precleared sample of step 4), then incubate with gentle agitation for 3 hours at 4 o C. 8) Wash the beads 4 times with Wash Buffer (+) (centrifuge the tube at 2,000 x g for 1 minute). 9) Add 400 uL of Master mix solution (Solution I: Solution II = 10 uL: 390 uL). Vortex for 10 seconds. 10) Add 250 uL of Solution III. Vortex for 10 seconds. 11) Centrifuge the tube at 2,000 x g for 2 minutes. 12) Transfer the supernatant to the tube containing 2 uL of Solution IV. 13) Add 600 uL of ice-cold 2-propanol and place at -20 o C for 20 minutes. Centrifuge the tube at 12,000 x g for 10 minutes. 14) Wash the pellet 2 times with 0.5 mL of ice-cold 70% Ethanol and dry up the pellet for 5-15 minutes. 15) Dissolve the pellets in nuclease-free water. RNA (ng) 54.0 527.0 Antibody Normal Rabbit IgG anti-STAU2 Average of the RNA Quantity (n=2) 133300.0 Total RNA 16) RNA was quantified with NanoDrop (Thermo Fisher Scientific Inc.) and the RNA quality was analyzed with Bioanalyzer (Agilent Technologies, Inc.). (Positive control for RNP Immunoprecipitation, Jurkat) 75 50 37 kDa Western blot analysis of STAU2 expression in 293T (1), HeLa (2), K562 (3), Jurkat (4) and SK-SY-5Y (5) using 1 2 3 4 5 RN013P. STAU2 SDS-PAGE & Western Blotting 1) Wash 1 x 10 7 cells 3 times with PBS and suspend them in 1 mL of Laemmli's sample buffer. 2) Boil the samples for 2 minutes and centrifuge. Load 10 uL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 3) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure. 4) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room.
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