The cells were lysed on ice for 5 min with equal volumes of RIP lysis (P0013B; Beyotime), followed by 10-min centrifugation at 14,000 rpm at 4°C, with the supernatant obtained. The binding of lncRNA MALAT1 and miR-204 to Ago2 protein and the binding of MYC mRNA to the IGF2BP2 protein were detected using RIP kits (Millipore, Billerica, MA, USA). In brief, 50 μL magnetic beads of each co-precipitation reaction system was re-suspended in 100 μL RIP Wash Buffer, followed by 6 h of incubation at 4°C and addition of 5 μg rabbit monoclonal antibody to Ago2 (ab186733, 1:50; Abcam) or mouse monoclonal antibody to IGF2BP2 (ab128175, 1:50; Abcam) for binding. The magnetic beads-antibody complex was then re-suspended in 900 μL RIP Wash Buffer followed by overnight incubation with 100 μL cell extract at 4°C. The samples were then placed onto a magnetic pedestal to collect the bead-protein complex. The samples were subsequently detached using protease K, after which the RNA content was extracted for qRT-PCR detection. The rabbit antibody against IgG (ab172730, 1:100; Abcam) was regarded as the NC.
Ab128175
Manufactured by Abcam
Sourced in United States
Ab128175 is a laboratory equipment product. It is a core component used in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab172730, by Abcam (3 mentions)
Ab208577, by Abcam (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (2 mentions)
Ab186733, by Abcam (1 mentions)
Rip kit, by Merck Group (1 mentions)
Ab264375, by Abcam (1 mentions)
Ab243880, by Abcam (1 mentions)
Ab221795, by Abcam (1 mentions)
Ab229700, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Anti mettl3, by Abcam (1 mentions)
Anti ctsl, by Proteintech (1 mentions)
Anti mettl14, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Bsa blocking solution, by Solarbio (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab76252, by Abcam (1 mentions)
Ab177477, by Abcam (1 mentions)
9 protocols using ab128175
1
RNA-Protein Interaction Profiling
2
m6A Regulators Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3
Immunohistochemical Evaluation of IGF2BP2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC was performed as described previously (11 (link)). The tissues were incubated with primary antibody against IGF2BP2 (ab128175, Abcam, 1:100) overnight in the refrigerator at 4°C. For negative controls, irrelevant primary antibodies were used. The corresponding secondary antibodies, conjugated to horseradish peroxidase (ab6721, Abcam, 1:1000), were incubated with the sections for 1 h at room temperature. After washing with PBS, the sections were incubated in horseradish enzyme-labeled chain avid in solution for 30 min at 37°C and washed again. The IHC score (0–9) was calculated by multiplying the intensity and the percentage scores (12 (link)).
4
Western Blot Analysis of RNA-Binding Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors (Beyotime Biotechnology), separated by electrophoresis and transferred to PVDF membranes (Bio‐Rad). After overnight incubation with primary Abs (anti‐METTL3 [1:1000 dilution, ab195352; Abcam], anti‐CTSL [1:500 dilution, 66,914‐1‐lg; Proteintech], anti‐insulin‐like growth factor 2 mRNA‐binding protein [IGF2BP2] [1:1000 dilution, ab128175; Abcam], anti‐METTL14 [1:1000 dilution, ab220030’ Abcam], anti‐RNA binding motif protein 15 [RBM15] [1:1000 dilution; PTM‐6163; Jingjie PTM BioLab], and anti‐β‐actin [1:5000 dilution; 4970S; Cell Signaling Technology]), the membranes were incubated with a HRP‐conjugated secondary Ab (1:5000 dilution, 7076S, 7074S; Cell Signaling Technology). The immunoreactions were visualized using the ECL Plus substrate (Merck Millipore). β‐Actin was used for normalization.
5
Western Blot Analysis of IGF2BP2, FEN1, and β-actin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues and cells were lysed in RIPA buffer (Beyotime, China). The lysates were denatured at 95°C for 5 min and then cooled down on ice. Then lysates were loaded on sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) (10%) and electrotransferred onto polyvinylidene difluoride (PVDF) membrane. After blocking with 5% BSA blocking solution (SW3015, solarbio) for 1 h at room temperature, PVDF membranes were blotted with primary antibody at 4°C for 12 h, then incubated with HRP-labeled secondary antibody (CST, USA) at room temperature for 2 h. The bands were visualized using Tanon 5200 (Tanon, China). Primary antibodies are as follows: mouse monoclonal antibody to IGF2BP2 (ab128175, Abcam), rabbit monoclonal antibody to FEN1 (ab109132), and mouse monoclonal antibody to β-actin (CST, USA).
6
Investigating RNA-binding Protein Functions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in this study were: anti-Myc tag (ab32, Abcam), anti-IGF2BP1 (ab290736, Abcam), anti-IGF2BP2 (ab128175, Abcam), anti-IGF2BP3 (ab177477, Abcam), anti-RBM15 (10587-1-AP, Proteintech), anti-DNMT1 (ab92314, Abcam), anti-DNMT3A (ab307503, Abcam), anti-DNMT3B(67,259, Cell Signaling Technology), anti-SEMA3F (SAB2107196, Sigma), anti-S9.6 (MABE1095, Sigma), anti-5-mC (ab214727, Abcam), anti-m6A (SAB5600251, Sigma), anti-YAP1(phosphor S127, ab76252, Abcam), anti-YAP1(66900-1-Ig, Proteintech), anti-LATS1(66569-1-Ig, Proteintech), anti-LATS2(20276-1-AP, Proteintech), anti-β-Actin(4970, Cell Signaling Technology). siRNAs against DNMT1 used were purchased from Ribobio Co.,Ltd (Guangzhou, China). pcDNA3-based vectors encoding wild-type, RRM domain mutant, KH domain mutant Myc-tagged IGF2BP1, IGF2BP2, IGF2BP3 were produced by Shanghai Yoche Biotechnology Co.,Ltd (Shanghai, China). The plasmids encoding RBM15 and SEMA3F (h-RBM15-pcDNA3.1-c-HA, M35-FLAG-SEMA3F) were obtained from Guangzhou FulenGen Co., Ltd (Guangzhou, China). DNA and RNA oligos synthesized by TsingKe Biotech Co., Ltd (Beijing, China) are listed in Supplementary Table S1 .
7
RIP Assay for m6A and IGF2BP2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17–700; Sigma-Aldrich) was used for the RIP assay following the manufacturer’s instructions. Cells were lysed and RNA–protein complexes were incubated with anti-m6A (ab208577; Abcam), anti-IGF2BP2 (ab128175; Abcam) or anti-IgG antibody (ab172730; Abcam) overnight at 4 °C and washed with RIP-wash buffer for 10 min at 4 °C and then RIP-lysis buffer for 5 min at 4 °C. The co-precipitated RNAs were purified using phenol:chloroform:isoamyl alcohol and subjected to RT-qPCR.
8
RNA Binding Protein Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA immunoprecipitation (RIP) assays were executed using the Magna RIP RNA Binding Protein Immunoprecipitation kit (Millipore). Cells were prepared using RIP lysis buffer and the RNA–protein complexes were conjugated with anti-IGF2BP2 (Abcam, ab128175, 1:30) or anti-IgG antibody (Abcam, ab172730, 1:30) overnight at 4 ℃ and washed with RIP wash buffer for 10 min at 4 ℃ and then the RIP lysis buffer for 5 min at 4 ℃. The coprecipitated RNAs were purified using phenol/chloroform/isoamyl alcohol and subjected to qRT-PCR.
9
m6A Regulation of NLRP3 mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
m6A modifications on NLRP3 mRNA were detected using the Magna RIP™Quad RNA-binding Protein immunoprecipitation Kit (17–704, Millipore, USA). Briefly, 200 mg of total RNA was enriched with anti-M6A antibody (ABE572, Merck, USA), anti-IGF2BP2 antibody (ab128175, Abcam, USA) or IgG-coupled protein A/G magnetic beads in 500 mL of 1×IP buffer supplemented with RNase inhibitors. Overnight at 4 ° C. The RNA of interest was immunoprecipitated with beads. One-tenth of each RNA fragment was stored as an input control for qPCR analysis with primers: NLRP3, 5′-CCATCAATGCTGCTTCGACA-3′ and 5′-GAGCTCAGAACCAATGCGAG-3′.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!