Cells (5 × 106) were lysed for 15 min on ice in a polysome lysis buffer containing 100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.0, 0.5% NP-40 detergent supplemented with fresh 1 mM dithiothreitol (DTT), 100 U/ml RNase Out (Invitrogen), 400 µM vanadyl-ribonucleoside complex (VRC) (New England Biolabs), and a protease inhibitor cocktail (Sigma). The cell lysate was further diluted (1:10) with NT2 buffer containing 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40 supplemented with fresh 200 U/ml RNase Out, 400 µM VRC, 1 mM DTT, 20 mM EDTA, and a protease inhibitor cocktail. The insoluble particles in the lysate were removed by centrifugation at 15,000 × g for 15 min at 4 °C. LIN28B antibody (1:75, Cell Signaling Technology), RPL26 antibody (1:50, Abcam) or control IgG was added to protein-A Sepharose beads (Sigma) that had been pre-incubated in 5% bovine serum albumin (BSA)–NT2 buffer for 1 h at 4 °C. After gentle rotation for 4 h at 4 °C, the beads were washed four times in cold NT2 buffer and added to the cell lysates (10 µl beads/ml lysate). Immunoprecipitation was performed by gentle rotation overnight at 4 °C. The immunoprecipitated complexes were washed four times in NT2 buffer and resuspended in 100 µl NT2 buffer containing 30 µg proteinase K (QIAGEN) to release the RNP complex. TRIzol reagent was used to extract RNA from the immunoprecipitation.
Lin28b antibody
Manufactured by Cell Signaling Technology
The LIN28B antibody is a research-use-only product that targets the LIN28B protein. LIN28B is a RNA-binding protein that plays a role in the regulation of cell differentiation and development. The antibody can be used to detect and study the expression and localization of LIN28B in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Igg pp64, by Merck Group (2 mentions)
Tri reagent, by Zymo Research (2 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Ribonucleoside vanadyl complex, by New England Biolabs (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Sepharose beads, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Cyclin b1 antibody, by Cell Signaling Technology (1 mentions)
Igf2bp1 antibody, by Cell Signaling Technology (1 mentions)
Beta actin hrp conjugated antibody, by Santa Cruz Biotechnology (1 mentions)
Cyclin d1 antibody, by Santa Cruz Biotechnology (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Normal rabbit igg, by Merck Group (1 mentions)
5 protocols using lin28b antibody
1
RNA immunoprecipitation and extraction protocol
2
Western Blot Analysis of Cell Cycle Regulators
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Total protein extracts were vortex and prepared in lysis buffer (1% Nonidet P-40, 50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 10% glycerol, protease and phosphatase inhibitors). The samples were separated on 12% polyacrylamide denaturing gels and transferred to PVDF membranes. Membranes were saturated for 1 h in 5% milk with 0.1% Tween 20-PBS (PBS-T) solution before incubating with the following antibodies: LIN28B antibody, IGF2BP-1 antibody, Cyclin B1 antibody, p21waf1 antibody from Cell Signaling Technologies (Danvers, MA) and Cyclin D1 antibody, beta-actin HRP conjugated antibody from Santa Cruz Biotechnology (Santa Cruz, CA).
3
In vivo RNA Immunoprecipitation of LIN28B
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In vivo RNA immunoprecipitation (RIP) was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit according to the manufacturer's protocol (Millipore). In brief, rat PFC was lysed and immunoprecipitated overnight at 4 °C with a LIN28B antibody (3 μg per immunoprecipitation; #5422, Cell Signaling Technology) or normal rabbit IgG (negative control, 3 μg per immunoprecipitation; Millipore) bound to protein A/G magnetic beads. Ten percent of the lysate was saved as RIP input and stored at −80 °C until starting RNA purification. After washing, the immunoprecipitate was digested by proteinase K followed by RNA purification. cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) followed by RT-PCR for pri-let-7 expression. The primer sequences are listed in Supplementary Table S1 .
4
RNA Immunoprecipitation in Differentiating HSPCs
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RNA immunoprecipitation (RIP) was carried out in differentiating HSPCs derived from newborns on day 7 of erythroid differentiation. Dynabeads were washed in blocking solution (PBS+0.5% BSA), and incubated overnight at 4 °C with end-to-end rotation in LIN28B antibodies (4196, Cell Signaling and A303–588A, Bethyl Labs) and IgG (PP64, Millipore) as control. Cells were lysed in NP40 lysis buffer, supplemented with Protease inhibitors, RNasin, and 1 mM DTT and pre-cleared with beads for 1 hour at 4 °C. After saving 1/50 volume for input, the rest of the pre-cleared lysate was incubated with the dynabeads-antibody complex for overnight at 4 °C with rotation. The beads-antibody-antigen complex was washed three times with low-salt buffer (5X PBS, 0.1% SDS, 0.5% Na-DOC and 0.5% NP-40) and then twice with high-salt buffer (1X PBS, 0.1% SDS, 0.5% Na-DOC and 0.5% NP-40). All washing steps were carried out at 4 oC, and the supernatant saved as flow through (FT). Elution buffer (50 mM HEPES, 0.1 M NaCl, 5 mM EDTA, 10 mM DTT, 0.5% Triton X-100, 10% glycerol, 1% SDS), freshly supplemented with RNase inhibitor was used. RNA extraction was carried out with TRI-Reagent (R2050–1-50, Zymo Research). RNA was quantified with Qubit, and qRT-PCR was subsequently carried out for LIN28B, HMGA2, GATA1, ALAS2, LDB1, KLF1 and LMO2 with primers listed in Supplementary Table 4 .
5
RNA Immunoprecipitation in Differentiating HSPCs
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