Immunoprecipitation
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.
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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).
Example 1
The ability of eyedrops to deliver Penl-XBIR3 in mice and rats was tested. Results are presented in
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
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 (
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).
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More about "Immunoprecipitation"
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.