RIP was performed using the EZMagna RIP Kit (Millipore, Billerica, MA, USA). The cell lysate was prepared using RNA lysis buffer. After centrifugation, the supernatant was incubated with magnetic beads coated with either an anti-IgG antibody (1:1000, ab172730, Abcam) or an anti-IGF2BP1 antibody (1:30, ab184305, Abcam). RIP lysate supernatant was used as input. Finally, the coprecipitated RNA was isolated, and the enrichment of circUHRF2 and DDX27 mRNA was determined by qRT-PCR experiment and normalized to the input.
Ab184305
Manufactured by Abcam
Sourced in United States
Ab184305 is a recombinant protein product. It is produced using a mammalian expression system.
Automatically generated - may contain errors
Lab products found in correlation
Ab195352, by Abcam (9 mentions)
Ab172730, by Abcam (4 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (4 mentions)
Ab8226, by Abcam (3 mentions)
Ab128958, by Abcam (3 mentions)
B15001, by Selleck Chemicals (3 mentions)
B14001, by Selleck Chemicals (3 mentions)
Ab181150, by Abcam (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ab188334, by Abcam (3 mentions)
C1053, by Applygen (3 mentions)
Ab177477, by Abcam (2 mentions)
Magna rip kit, by Merck Group (2 mentions)
Ab225697, by Abcam (2 mentions)
Ab208577, by Abcam (2 mentions)
Nbp1 46846, by Novus Biologicals (2 mentions)
Ab51608, by Abcam (2 mentions)
Nano zoomer digital pathology biopsy scanner, by Hamamatsu Photonics (2 mentions)
3 3 diaminobenzidine (dab), by ZSGB-BIO (2 mentions)
Pv 9000, by ZSGB-BIO (2 mentions)
25 protocols using ab184305
1
RNA Immunoprecipitation for circUHRF2 and DDX27
2
Protein Expression Analysis in DEN-Treated Cells
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SMMC-7721 or MHCC97h cells were treated with DEN (4 mM) or not, lysed, and then subjected to SDS-PAGE and immunoblotting, as previously described [24 (link)]. Primary antibodies against NONO (1:500; ab70335, Abcam, USA), ACLY (1:500; ab40793, Abcam, USA), Insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) (1:500; ab184305, Abcam, USA), Splicing factor proline and glutamine rich (SFPQ) (1:500; ab11825, Abcam, USA), Flag (1:1000; ab1162, Abcam, USA), Myc (1:1000; ab32072, Abcam, USA), and GAPDH (1:1000; ab181602, Abcam, USA) were used.
3
Immunoprecipitation and Western Blot Analysis
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Total protein was extracted and the target protein was detected as described previously.33 For immunoprecipitation (IP), the lysate was precleared by protein A/G‐agarose, and IP was carried out by incubating the supernatants with antibodies against NCAPH or β‐catenin and protein A/G‐agarose overnight at 4°C. After centrifugation, the complexes were washed and resuspended. The targeting proteins were detected by Western blotting. Rabbit IgG isotype was used as a negative control. All antibody information was listed as follows: NCAPH (PA5‐80842, Invitrogen), β‐actin (ab8226, Abcam), FOXP3 (ab215206, Abcam), FUS (PA5‐52610, Invitrogen), METTL3 (ab195352, Abcam), YTHDC1 (ab259990, Abcam), IGF2BP1 (ab184305, Abcam), IGF2BP2 (ab248279, Abcam), IGF2BP3 (ab248279, Abcam), β‐catenin (ab6301, Abcam), PD‐L1 (ab213480, Abcam), Granzyme‐B (MA1‐80734, Invitrogen), Perforin (14‐9993‐82, Invitrogen).
4
Quantifying ALKBH5 and IGF2BP1 Interactions
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An EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, 17–701) was used according to the manufacturer’s instructions. Briefly, MSCs were lysed and incubated with magnetic beads conjugated with anti-ALKBH5 (Proteintech, 16837–1-AP, 1:1,00), anti-IGF2BP1 (Abcam, ab184305, 1:1,00) or negative control IgG (Abcam, ab172730, 1:100). The immunoprecipitated RNAs were purified and extracted. The obtained RNAs and the input RNAs were subjected to qPCR followed by electrophoresis to detect the CYP1B1 mRNA level.
5
Comprehensive Protein Expression Analysis in Stem Cell Research
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Antibodies against octamer-binding transcription factor 4 (1:1000, Oct-4; ab200834, abcam), Brachyury (1:1000, sc-166962, Santa Cruz Biotechnology), GATA-binding protein 4 (1:1000, GATA4; sc-25310, Santa Cruz Biotechnology), SRY box transcription factor 17 (1:1000, Sox17; AF1924, R&D system), neuronal differentiation 1 (1:1000, NeuroD1; ab205300, abcam), XRCC5 (1:2000, AF5819, R&D system), IGF2BP1 (1:1000, ab184305, abcam), RBBP4 (1:1000, MAB7416, R&D system), NPM1 (1:1000, sc-271737, Santa Cruz Biotechnology), PCNA (1:1000, sc-56, Santa Cruz Biotechnology), GNAI2 (1:1000, ab137050, abcam), PHB2 (1:1000, sc-133084, Santa Cruz Biotechnology), LAMB1 (1:1000, sc-17810, Santa Cruz Biotechnology), LAMC1 (1:1000, sc-17751, Santa Cruz Biotechnology), β-catenin (1:1000, sc-7963, Santa Cruz Biotechnology), AKT (1:2000, #4691, Cell Signaling Technology), Jun amino-terminal kinases (JNK) (1:1000, #9252, Cell Signaling Technology), phosphorylated (p)-JNK (1:1000, #9255, Cell Signaling Technology), p38 (1:1000, sc-81621, Santa Cruz Biotechnology), p-p38 (1:1000, #9216, Cell Signaling Technology), TGF-β (1:1000, #3711, Cell Signaling Technology), CTGF (1:1000, sc-385970, Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; (1:3000, sc-47724, Santa Cruz Biotechnology) were employed for the western blot analysis.
6
RIP Assay for m6A RNA Binding Proteins
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RIP experiments were performed using the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) and the m6A antibody (Abcam, Cambridge, MA, USA) following the manufacturer’s protocol.
Briefly, cells (1×107) were lysed with ice-cold RIP Lysis Buffer. Collect and store the cell lysate at -80 °C. Prepare the magnetic bead-antibody complex with 5 μg of the above-mentioned target antibody or control IgG and 50 μl of protein A/G magnetic beads, rotating at room temperature for 30 min. Take an equal volume of cell lysate and incubate the magnetic bead-antibody complex with rotation at 4 °C overnight so that the antibody can fully contact and bind to the protein. At the same time,10 μl cell lysate was extracted and used as input. Next day, RNA was extracted and purified with the prepared proteinase K buffer. Acquired RNA was used as a template to synthesize the corresponding cDNA.
Co-precipitated RNAs used for the first strand cDNA synthesis with the Reverse Transcription System (Roche) following the manufacturer’s protocol. Acquired cDNA was then analyzed by RT-qPCR. The information of IGF2BP1/2/3 antibody are ab184305, ab117809 and ab177477 (Abcam, Cambridge, MA, USA).
Briefly, cells (1×107) were lysed with ice-cold RIP Lysis Buffer. Collect and store the cell lysate at -80 °C. Prepare the magnetic bead-antibody complex with 5 μg of the above-mentioned target antibody or control IgG and 50 μl of protein A/G magnetic beads, rotating at room temperature for 30 min. Take an equal volume of cell lysate and incubate the magnetic bead-antibody complex with rotation at 4 °C overnight so that the antibody can fully contact and bind to the protein. At the same time,10 μl cell lysate was extracted and used as input. Next day, RNA was extracted and purified with the prepared proteinase K buffer. Acquired RNA was used as a template to synthesize the corresponding cDNA.
Co-precipitated RNAs used for the first strand cDNA synthesis with the Reverse Transcription System (Roche) following the manufacturer’s protocol. Acquired cDNA was then analyzed by RT-qPCR. The information of IGF2BP1/2/3 antibody are ab184305, ab117809 and ab177477 (Abcam, Cambridge, MA, USA).
7
RNA-Binding Protein Immunoprecipitation
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Cells were lysed with a complete RIP lysis buffer (Millipore, Burlington, MA, USA). Cell extract was incubated with immunoglobulin G (IgG) (1:50, ab172730; Abcam, Waltham, MA, USA) and IGF2BP1 (1:50, ab184305; Abcam) antibody at 4°C overnight. RNA samples were purified by phenol chloroform extraction, followed by quantitative real-time polymerase chain reaction (qRT-PCR) to determine MCF2L-AS1 transcripts enrichment.
8
RNA-Binding Protein Immunoprecipitation
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Cultured cells were cross-linked at 200 mJ/cm2 using 254 nm UV light with an Ultraviolet Crosslinker (UVP) before being lysed as previously described 23 (link). RIP assays were conducted using the Magna RIP Kit (17-700, Millipore, MA), with antibodies specific for FUS (11570-1-AP, Proteintech), IGF2BP1 (ab184305, abcam), IGF2BP2 (11601-1-AP, Proteintech), IGF2BP3 (ab225697, abcam), or DYKDDDDK Tag (20543-1-AP, Proteintech). The precipitated RNAs were then analyzed by qRT-PCR. The relative amount of immunoprecipitated RNA in each sample was represented as that of negative (IgG) sample.
9
Protein Extraction and Western Blot Analysis
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Proteins were extracted using radioimmunoprecipitation assay buffer (ab288006; Abcam) and quantified by BCA Protein Assay Kit (PICPI23223; Thermo Fisher Scientific). 40 μg of protein was separated by sodium dodecyl-sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. After blocking, membranes were incubated with antibodies against PIGT (1:500; 16,906–1-AP; Proteintech Group, Inc., Rosemont, IL, USA), GLUT1 (1:30,000; ab115730; Abcam), METTL3 (1:1000; ab195352; Abcam), METTL14 (1:500; ab220030; Abcam), WTAP (1:1000; ab195380; Abcam), IGF2BP1 (1:1000; ab184305; Abcam), IGF2BP2 (1:1000; ab129071; Abcam), IGF2BP3 (1:1000; ab177477; Abcam), and β-actin (1:1000; ab8226; Abcam) at room temperature for 1 h. Secondary antibodies were labeled with HRP (1:1000; A0208, A0216; Beyotime Biotechnology, Shanghai, China). Specific signals were visualized using an enhanced chemiluminescence substrate kit (P0018F, Beyotime Biotechnology).
10
Investigating m6A-Binding Protein Interactions
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RNA immunoprecipitation assay (RIP)/methylated RNA immunoprecipitation (MeRIP)‐qPCR was carried out using the Magna RIP RNA‐Binding Protein Immunoprecipitation Kit (#17–700; Millipore Sigma) according to the manufacturer's instructions. Anti‐METTL3 (ab195352; Abcam), anti‐IGF2BP1 (ab184305; Abcam), anti‐IGF2BP2 (11601‐1‐AP; Proteintech), anti‐IGF2BP3 (14642‐1‐AP; Proteintech), anti‐m6A (202,003; Synaptic Systems), and rabbit IgG control Abs were used. CTSL mRNA levels were quantified by RT‐qPCR, with normalization to the amount of input RNA. The specificity of RNA–protein interactions was tested by IgG.
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