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Immunoprecipitation

Immunoprecipitation is a powerful technique used to isolate and study specific proteins and their interactions within a complex biological sample.
This method involves the use of antibodies to selectively bind and precipitate target proteins, allowing researchers to investigate their structure, function, and associations with other biomolecules.
Immunoprecipitation plays a crucial role in proteomics, cell signaling, and protein-protein interaction studies, enabling researchers to gain insights into the complex dynamics of cellular processes.
By combining immunoprecipitation with advanced analytical tools, such as mass spectrometry, researchers can identify and characterize the proteins of interest, leading to a deeper understanding of biological systems and the development of new therapeutic interventions.
The PubCompare.ai platfrom can helmp optimize your immunoprecipitation research, providing access to the best protocols from literature, pre-prints, and patents, ensuring reproducible and accurate results.

Most cited protocols related to «Immunoprecipitation»

Cells were solubilized with lysis buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25 mM pAPMSF] and centrifuged at 12,000 × g for 20 min. For co-immunoprecipitation assay, cells were sonicated briefly in lysis buffer before centrifugation. The lysates were subjected to immunoprecipitation, SDS-PAGE, and Western blot analyses as described2 (link).
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Publication 2017
Aprotinin Biological Assay Buffers Cells Centrifugation Co-Immunoprecipitation Edetic Acid Immunoprecipitation leupeptin Nonidet P-40 SDS-PAGE Sodium Chloride Tromethamine Western Blot
(See Supplementary Protocol 1 for detailed Standard Operating Procedures for ENCODE-style eCLIP experiments, including oligonucleotide sequences, catalog numbers for all reagents, and specific details for eCLIP experiments). RNA binding protein (RBP)-RNA interactions were stabilized with UV crosslinking (254 nm, 400 mJ/cm2), followed by lysis in iCLIP lysis buffer, limited digestion with RNase I (Ambion), immunoprecipitation of RBP-RNA complexes with a specific primary antibody of interest using magnetic beads with pre-coupled secondary antibody (typically M-280 Sheep Anti-Rabbit IgG Dynabeads, ThermoFisher Scientific 11204D), and stringent washes. After dephosphorylation with FastAP (ThermoFisher) and T4 PNK (NEB), a barcoded RNA adapter was ligated to the 3′ end (T4 RNA Ligase, NEB) (at this step, multiple replicates of the same RBP, or potentially RBPs of similar size and bound RNA amount, can be uniquely barcoded and pooled after ligation to simplify downstream steps - see Supplementary Fig. 2a). Ligations were performed on-bead (to allow washing away unincorporated adapter) in high concentration of PEG8000, which improves ligation efficiency to > 90%. Samples were then run on standard protein gels and transferred to nitrocellulose membranes, and a region 75 kDa (~150 nt of RNA) above the protein size was isolated and proteinase K (NEB) treated to isolate RNA. RNA was reverse transcribed with AffinityScript (Agilent), and treated with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides. A second DNA adapter (containing a random-mer of 5 (N5) or 10 (N10) random bases at the 5′ end) was then ligated to the cDNA fragment 3′ end (T4 RNA Ligase, NEB), performed with high concentration of PEG8000 (to improve ligation efficiency) and DMSO (to decrease inhibition of ligation due to secondary structure). After cleanup (Dynabeads MyOne Silane, ThermoFisher), an aliquot of each sample was first subjected to qPCR (to identify the proper number of PCR cycles), and then the remainder was PCR amplified (Q5, NEB) and size selected via agarose gel electrophoresis. Samples were sequenced on the Illumina HiSeq 2500 or 4000 platform as two Paired End 50bp (for N5) or 55bp (for N10) reads. All analyses were performed using identical antibody lots for RBFOX2 (A300-864A lot 002, Bethyl), SLBP (RN045P lot 001, MBL International), and IgG Isotype Control (02-6102 lot 32013, Thermo Fisher Scientific). SLBP experiments were performed with 20×106 cells and 10 ug of primary antibody; RBFOX2 experiments were performed with 20×106 cells and 10 ug (eCLIP Rep1 and Rep2) or 10×106 cells and 5 ug (RNase I variation experiments). All experiments in K562 and HepG2 cells were performed with 20×106 cells and 10 ug of indicated primary antibody (Supplementary Table 2). Antibody validation documentation (including Western images of immunoprecipitation and shRNA knockdown19 (link)) are available at http://www.encodeproject.org/. Additional experiments performed in K562 and HepG2 cells in which the antibody failed to successfully immunoprecipitate the targeted RBP were excluded from analysis. 293T cells were obtained from Clontech (Lenti-X 293T cell line). K562 and HepG2 cells were purchased from ATCC, and were not independently verified. Cells were routinely tested for mycoplasma using MycoAlert PLUS (Lonza).
Publication 2016
anti-IgG Buffers Cell Lines Cells Digestion DNA, Complementary Domestic Sheep Electrophoresis, Agar Gel Endopeptidase K Gels HEK293 Cells Hep G2 Cells Immunoglobulin Isotypes Immunoglobulins Immunoprecipitation Ligation M 280 Mycoplasma Nitrocellulose Oligonucleotides polyethylene glycol 8000 Proteins Psychological Inhibition Rabbits Ribonuclease, Pancreatic RNA-Binding Proteins RNA Ligase (ATP) Short Hairpin RNA Silanes Sulfoxide, Dimethyl Tissue, Membrane
See Supplementary Methods for source of prostate tissues and cell lines, nucleic acid isolation, exome and transcriptome sequencing and data analysis, mutation validation by Sanger sequencing, aCGH and DNA microarray expression profiling, ETS2 in vitro experiments, AR interactor immunoprecipitation, western blotting, and siRNA experiments, FOXA1 screening and in vitro and in vivo experiments.
Publication 2012
Cell Lines Exome FOXA1 protein, human Immunoprecipitation isolation Microarray Analysis Mutation Nucleic Acids Prostate RNA, Small Interfering Tissues

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Publication 2012
Brain Buffers Genome Immunoprecipitation Mice, House Ribosomal RNA RNA, Messenger
Data used in this study are summarized in Supplementary Table 1 online. The NRSF ChIP-chip data (GEO accession #: GSE8489) were obtained by analyzing the bound DNA fragments in Jurkat cells with Affymetrix Human Tiling 2.0R arrays. Two independent ChIP samples and two mock IPs were profiled. The NRSF ChIP-seq data were collected from a previous study4 (link). In that study, DNA fragments bound by NRSF in Jurkat cells were sequenced with the next generation sequencer made by Illumina/Solexa. These experiments involved sequencing a ChIP'd sample as well as a negative control sample generated from reverse-crosslinked genomic DNA that had not undergone immunoprecipitation. The Oct4 and Nanog ChIP-seq data were collected from [10].
Publication 2008
ChIP-Chip Chromatin Immunoprecipitation Sequencing DNA Chips Genome Homo sapiens Immunoprecipitation Jurkat Cells POU5F1 protein, human

Most recents protocols related to «Immunoprecipitation»

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Example 3

To further confirm that the PEDV S protein contains the epitope recognized by the present scFv, an immunoprecipitation combined pull-down assay and SEC analysis were performed in this example. For the immunoprecipitation assay, the purified trimeric PEDV S glycoprotein, which harbored a V5 tag and a His6 tag, was incubated with the recombinant scFv for 3 hours and size-filtrated by centrifugation with a 100 kDa molecular weight cut-off (MWCO) spin column. In the absence of the PEDV S protein, all scFv passed through the 100 kDa MWCO spin column in the control group, and no protein band was detected in the respective lane of the SDS-PAGE (data not shown). The addition of the PEDV S protein retained the scFv after the 100 kDa MWCO filtration (data not shown). The SEC analysis of the PEDV S protein with excess scFv showed a similar elution volume as that without scFv, potentially due to the relatively small change in molecular size of the PEDV S protein when bound to the scFv (data not shown). To ascertain that the scFv was indeed co-eluted with the PEDV S protein during the SEC, the elution fractions corresponding to the PEDV S protein were analyzed by western blotting. The data confirmed the binding between the present scFv and PEDV S protein (data not shown).

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Patent 2024
Biological Assay Centrifugation Epitopes Filtration Glycoproteins his6 tag Immunoprecipitation Lanugo Porcine epidemic diarrhea virus Proteins SDS-PAGE spike protein, SARS-CoV-2

Example 1

The ability of eyedrops to deliver Penl-XBIR3 in mice and rats was tested. Results are presented in FIG. 2 and FIG. 3.

In mice, Penl-XBIR3 (10 μg) eyedrops were applied, then the animals were sacrificed at the indicated times. In rabbits, 200 μg Penl-XBIR3 eyedrops or a saline vehicle were administered BID for 4.5 days. The final dose given 5 h prior to harvest of retinas. Plasma from rabbits obtained at baseline and harvest.

Retinal lysates were immunoprecipitated with XIAP, followed by western blotting for anti-His. XBIR3 contains a His tag, so uptake of XBIR3 is detectable using anti-His. Blots for the mouse and rabbit samples, along with graphs quantifying the results, are presented in FIG. 2. XBIT3 uptake was observed in both mouse and rabbit samples. Uptake in the mouse samples was detected by 1 h and maintained through 24 h. In rabbit there was significant XBIR3 in retina at 5d.

Baseline and post-treatment plasma from rabbits was analyzed by immunoprecipitation with XIAP followed by western blot with anti-His. A Ponceau protein stain was used to show input protein amounts. XBIR3 was not detected in rabbit plasma (FIG. 3), indicating that it remains localized in the eye.

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Patent 2024
Animals Eye Drops Immunoprecipitation Mice, House Oryctolagus cuniculus Plasma Proteins Rabbits Rattus Retina Saline Solution Stains Western Blotting
The RIP assays were conducted using the TAF15 (Abcam) and IgG (Abcam) control -specific antibodies, and the RNA Immunoprecipitation Kit (Geneseed). Expression levels of RNA and target protein in the samples were assessed by qRT-PCR and western blot, respectively.
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Publication 2023
Antibodies Biological Assay Immunoprecipitation Protein Targeting, Cellular TAF15 protein, human Transcription, Genetic Western Blotting
In detail, cells were collected and lysed in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholine, and 1 mM NaF), and the protein concentration was measured using a BCA protein assay kit (Pierce Biotechnology). Proteins were separated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After 2 h, the membranes were blocked with 5% skim milk and incubated with the primary antibodies at 4 °C overnight. After that, the membranes were washed with TBST for three times and incubated with HRP-labeled goat anti-rabbit antibody [Cell Signaling Technology (CST), USA] for 0.5 h. Finally, the immunoreactive signals were detected using the ECL detection system (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, IL, USA). The following primary antibodies were applied: rabbit anti-HIF1α (36169S, CST, USA), rabbit anti-P-p65 (3033S, CST, USA), rabbit anti-GPR116 (ab136262, Abcam, Shanghai, China), rabbit anti-p65 (8242S, CST, USA), rabbit anti-β-actin (4970 L, CST, USA), rabbit anti-GNA11 (ab153951, abcam, UK).
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Publication 2023
Actins Antibodies Antibodies, Anti-Idiotypic Biological Assay Buffers Cells Edetic Acid Goat HIF1A protein, human Hypersensitivity Immunoprecipitation Milk, Cow's Nonidet P-40 polyacrylamide gels polyvinylidene fluoride Proteins Rabbits Sodium Sodium Chloride Tissue, Membrane Tromethamine
Cell lysate preparation, SDS-PAGE, and Western blotting were carried out according to standard protocol. Proteins were harvested in cell lysis buffer supplemented with proteinase inhibitor cocktail (P8340; Sigma-Aldrich) and phosphatase inhibitor cocktail 2 (P5726; Sigma-Aldrich). Antigen detection was performed using antibodies directed against c-Src (rabbit anti-mouse/human antibody; #2109; Cell Signaling), Ctsk (mouse anti-mouse/human antibody; sc-48353; Santa Cruz), Rho (mouse anti-mouse/human antibody; #05-778; Millipore), galectin-3 (mouse anti-mouse/human antibody; ab2785; Abcam; epitopes mapped within the N-terminal region), Lrp1 (mouse anti-mouse antibody; MABN1796; Millipore), Mmp9 (rabbit anti-mouse antibody; ab38898; Abcam), Mmp14 (rabbit anti-mouse antibody; ab53712; Abcam), OXPHOS (rabbit anti-mouse antibody; ab110413; Abcam), vinculin (mouse anti-mouse antibody; V9131; Sigma-Aldrich), β3 integrin (rabbit anti-mouse antibody; #4702; Cell Signaling), or β-actin (rabbit anti-mouse antibody; #4970; Cell Signaling). Goat anti-rabbit IgG horseradish peroxidase (#65-6120; Thermofisher Scientific) or goat anti-mouse IgG horseradish peroxidase (#32430; Thermofisher Scientific) were used as secondary antibody. Bound primary antibodies (diluted to 1:1,000) were detected with horseradish peroxidase–conjugated species-specific secondary antibodies (Santa Cruz; diluted to 1:2,000) using the Super Signal Pico system (Thermo Fisher Scientific).
For immunoprecipitation analysis, cells were solubilized in IP Lysis Buffer (#87788; Thermo Fisher Scientific) supplemented with complete protease inhibitor cocktail (Roche). Immunoprecipitation was performed by incubation with a mouse monoclonal IgG (#5415; Cell Signaling) or anti–galectin-3 antibody (sc-32790; Santa Cruz) followed by the addition of Protein A/G Magnetic Beads (#88803; Thermo Fisher Scientific). Immune complexes were separated by electrophoresis followed by blotting with antibodies directed against Lrp1 (MABN1796; Millipore) and galectin-3 (ab2785; Abcam).
Publication 2023
Actins anti-IgG Antibodies Antibodies, Anti-Idiotypic Antigens Buffers Cells Complex, Immune CTSK protein, human Electrophoresis Epitopes G-substrate Galectin 3 Goat GTP-Binding Proteins Homo sapiens Horseradish Peroxidase Immunoglobulins Immunoprecipitation Integrin beta3 MMP9 protein, human MMP14 protein, human Mus Protease Inhibitors protein phosphatase inhibitor-2 Proteins Rabbits SDS-PAGE Staphylococcal Protein A Vinculin

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The Magna RIP RNA-Binding Protein Immunoprecipitation Kit is a laboratory equipment product designed for the isolation and identification of RNA-binding proteins. It provides a standardized protocol and necessary components to perform RNA-protein immunoprecipitation experiments.
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Protease inhibitor cocktail is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It is commonly used in protein extraction and purification procedures to prevent protein degradation.
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More about "Immunoprecipitation"

Immunoprecipitation (IP) is a powerful analytical technique used to isolate and study specific proteins and their interactions within complex biological samples.
This method leverages the selective binding of antibodies to target proteins, enabling researchers to investigate their structure, function, and associations with other biomolecules.
IP plays a crucial role in proteomics, cell signaling, and protein-protein interaction studies, providing insights into the dynamic mechanisms of cellular processes.
By combining IP with advanced analytical tools, such as mass spectrometry, researchers can identify and characterize the proteins of interest, leading to a deeper understanding of biological systems and the development of new therapeutic interventions.
The Magna RIP RNA-Binding Protein Immunoprecipitation Kit, for example, allows for the isolation and study of RNA-binding proteins and their associated RNAs, while Protease inhibitor cocktail can be used to preserve the integrity of the target proteins during the IP process.
PVDF membranes and Dynabeads Protein G are commonly utilized in IP experiments to facilitate the separation and detection of the captured proteins.
Additionally, the Anti-FLAG M2 affinity gel can be employed to selectively purify proteins tagged with the FLAG epitope.
Lipofectamine 2000 is a transfection reagent that can be used to introduce expression vectors for the proteins of interest, enabling their subsequent immunoprecipitation.
The PubCompare.ai platform can help optimize your IP research by providing access to the best protocols from literature, pre-prints, and patents, ensuring reproducible and accurate results.
By leveraging the power of Dynabeads, Protein G Dynabeads, and the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit, you can unlock the full potential of your IP-based investigations and gain valuable insights into the complex dynamics of cellular machinery.