The RIP assay was performed using an RNA Immunoprecipitation Kit (BersinBio, Guangzhou, China). The cell lysate was incubated with RIP buffer containing magnetic beads conjugated with Argonaute-2 (AGO2) or IgG control antibody (Abcam, Waltham, MA, USA). The expression of circPTPN22 in immunoprecipitates was determined by real-time PCR.
Control igg antibody
Manufactured by Abcam
Sourced in United States
Control IgG antibody is a non-specific immunoglobulin G (IgG) that can be used as a negative control in various immunoassays and experiments. It serves as a baseline reference to distinguish specific antibody signals from non-specific background.
Automatically generated - may contain errors
Lab products found in correlation
Ez magna rip kit, by Merck Group (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Rna immunoprecipitation kit, by BersinBio (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Anti il 6 antibody, by Merck Group (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Nf κb, by Abcam (1 mentions)
Chip assay kit, by Cell Signaling Technology (1 mentions)
880 airyscan confocal microscope, by Zeiss (1 mentions)
Anti il1r1, by R&D Systems (1 mentions)
Anti ago2, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Ago2 antibody, by Abcam (1 mentions)
Ago2 antibody, by Thermo Fisher Scientific (1 mentions)
Rip lysis buffer, by Merck Group (1 mentions)
Protein g magnetic beads, by Merck Group (1 mentions)
Ab2768, by Abcam (1 mentions)
Phospho stat3 y705, by Abcam (1 mentions)
Total stat3, by Santa Cruz Biotechnology (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
12 protocols using control igg antibody
1
Profiling circPTPN22 expression by RIP
2
Rapamycin Modulates IGFBP7-Induced Osteoblastic Reprogramming
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Fibroblasts at passage 10 were cultured as described above. Cells were washed and fresh medium containing with IGFBP7 (1 μg/mL) with in water or rapamycin (500 nM, Sigma‐Aldrich, Saint Louis) and 0.1% DMSO was added to cells to examine the effect of rapamycin on IGFBP7‐induced osteoblastic reprogramming P7. For the study of IL‐6 signaling, IgG control antibody (50 μg/mL; Abcam, Cambridge, MA) or anti IL‐6 antibody (5ug/ml; I7901, Sigma‐Aldrich, Saint Louis) was added to cells culturing in media containing IGFBP7 (1 μg/mL). Cells were grown at 37°C with 5% CO2, and the medium was changed every 3 days. Cells were harvested for gene expression analysis at day 4 and 14, and for Alizarin red staining at day 28.
3
RIP Assay of TLR2, TLR4, NF-κB
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RIP assay was carried out according to the protocol of Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA). Cell lysate was incubated with RIP immunoprecipitation buffer containing magnetic beads, which could conjugate with TLR2, TLR4 (Abcam, Cambridge, USA), NF-κB (CST, USA), and IgG control antibody (Abcam, Cambridge, USA). HIX003209 RNA level in immunoprecipitates was determined by real-time PCR.
4
ChIP Assay Protocol for KDM5A
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ChIP assays were conducted with a ChIP Assay Kit (Cell Signaling Technology [CST], Danvers, MA, USA). In brief, Leuk-1 cells (5 × 108) were fixed with a final concentration of 1% formaldehyde, crosslinked, and sonicated. The KDM5A antibody (10 μg/mL, Abcam), IgG control antibody (2 μg/mL, Abcam) was added to sonicated lysates and incubated overnight at 4°C, then incubated with a Protein A/G beads mixture (1:1 ratio, CST) for another >7 h at 4°C. Chromatin DNA was eluted, reverse crosslinked, and recovered using a ChIP Assay Kit (CST). Input DNA and immunoprecipitated DNA were analyzed by qPCR using promoter DNA-specific primers listed in Table S4 .
5
Immunofluorescence Staining of NTF Cells
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NTF were seeded on glass coverslips at 1 x104 cells in 24‐well plates and incubated overnight before being fixed and permeabilised using 2% paraformaldehyde and 0.1% Triton‐X‐100 for 15 min. After blocking with bovine serum albumin for 30 min, cells were incubated with 5 μg/mL of either anti‐IL‐1R1 (R&D Systems, clone 73,229), HLA class I (Sigma‐Aldrich, clone W6/32) or IgG control antibody (Abcam) at 4 °C overnight. After washing with PBS, cells were incubated with biotin‐conjugated goat anti‐mouse antibody (Abcam) for 30 min then with phycoerythrin‐conjugated streptavidin for 20 min at room temperature. after washing and mounting with ProLong™ Diamond anti‐fade containing 4′,6‐Diamidine‐2′‐phenylindole dihydrochloride (DAPI), cells were imaged using a Zeiss880 AiryScan confocal microscope.
6
Quantifying let-7a-5p Binding in Cardiomyocytes
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Let-7a-5p and let-7a-5p mutants were transfected into cardiomyocytes for 48 h. The cells were lysed in RIPA lysis buffer (Cell Signaling Technology, Inc.) for 30 min on ice and incubated with anti-AGO2 (1:100; cat. no. ab186733; Abcam) or control IgG antibody-(1:100; cat. no. 3900s; Cell Signaling Technology, Inc.) coupled Sepharose beads for 4 h at 4°C under rotation. The bound RNAs were purified by washing with lysis buffer (Wuhan Boster Biological Technology, Ltd.). The purified RNA was used for RT-qPCR according to the aforementioned protocol. GAPDH was used as a negative control.
7
RIP Assay Verifies miR-214-5p Binding
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RIP was used to verify the binding relationship between miR-214-5p and PSMA3-AS1 or PD-L1 [22 (link)]. RIP assay was conducted with an EZMagna RIP kit (Merck KGaA). Cell lysates were treated with magnetic beads conjugated to Ago2 antibody (Abcam) or control IgG antibody (Abcam) in RIP buffer. All precipitated RNAs were examined by RT-qPCR.
8
Ago2-Mediated RNA Immunoprecipitation
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The RIP assay was performed using the EZ-Magna RIP Kit (MilliporeSigma) following the manufacturer's recommendations. In brief, C28/I2 cells were lysed in RIP lysis buffer (MilliporeSigma) containing protease inhibitor cocktail and RNase inhibitor for 5 min of incubation on ice followed by centrifugation at 10,000 x g for 10 min at 4˚C. Cell lysate was incubated with RIP buffer containing Protein G magnetic beads (cat. no. 88848; Thermo Fisher Scientific, Inc.) coated with Ago2 antibody (1:50; cat. no. ab186733; Abcam) or the control IgG antibody (1:1,000; cat. no. ab6702; Abcam) overnight at 4˚C according to the manufacturer's recommendations. Next, the magnetic beads were washed three times using RIP wash buffer and treated with proteinase K (Thermo Fisher Scientific, Inc.) at 55˚C for 30 min. Finally, the co-precipitated RNAs were measured using RT-qPCR.
9
Immunoprecipitation of Ago2-Bound RNAs
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Cell extracts were collected from RIP lysis buffer for incubation for 4 h in RIP buffer adding the magnetic beads coated human Ago2 antibody or control IgG antibody (Abcam, Cambridge, MA). After elution, precipitated RNAs were extracted and analyzed.
10
Chromatin Immunoprecipitation of MYC Protein
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Cells were fixed for 20 minutes for forming the DNA and protein cross‐linking, then fragmented by ultrasonic and immunoprecipitated with anti‐MYC antibody or control IgG antibody (Abcam). 30 μL magnetic beads were then added for 2 hours, and precipitated DNA was purified for qRT‐PCR.
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