eluting epitope tag precipitated proteins and immunoblotting for protein-protein interaction studies

使用免疫共沉淀法对 HEK-293 细胞进行蛋白质相互作用分析

Proteins play a major role in all cellular functions, including information processing, metabolism, transport, decision-making, and structural organization. Proteins mediate their functions by interacting physically with other molecules. Protein-protein interactions (PPIs) are important for mediating cellular functions, such as mediating signal transduction, sensing the environment, converting energy into physical movement, regulating the activity of metabolic and signaling enzymes, and maintaining cellular organization1. Thus, PPIs can be used to elucidate unknown functions2. Methods for detecting PPIs can be classified into three types: in vitro, in vivo, and in silico. Co-immunoprecipitation (co-IP), affinity chromatography, tandem affinity purification, protein arrays, phage display, protein fragment complementation, X-ray crystallography, and nuclear magnetic resonance spectroscopy have been used for in vitro PPI detection3. Among these methods, co-IP is widely used because of its simplicity.
The fusion tag FLAG consists of eight amino acids (AspTyrLysAspAspAspAspLys: DYKDDDDK), including an enterokinase cleavage site, and was specifically designed for immunoaffinity chromatography4. DYKDDDDK-tagged proteins are recognized and captured using an anti-DYKDDDDK antibody. Therefore, they are efficiently pulled down using DYKDDDDK binding agarose beads5 to confirm their binding to specific proteins in a simple manner. Immunoprecipitation can be performed in a variety of cells, and a wide range of PPIs can be confirmed using antibodies against the protein of interest. Immunoprecipitation and peptide elution with anti-DYKDDDDK agarose beads have been previously reported5.
Here, we provide a simple immunoprecipitation method in which a plasmid encoding a DYKDDDDK-tagged protein is transiently introduced into HEK-293 cells to confirm the association of two proteins of interest. Certain DYKDDDDK antibodies can bind to both the N-terminus and C-terminus of the fusion proteins but not others6. Therefore, to avoid confusion, the antibody that recognizes the tag fused to both N- and C-terminus should be chosen. When inserting an epitope tag, it may be possible to avoid conformational changes in the protein by inserting 3 to 12 base pairs between the epitope tag and the target protein. However, the inserted sequence should be a base pair in multiples of 3 to avoid frameshift.

作者聚焦：揭示棕色和米色脂肪细胞调节的分子机制

使用抗表位标签亲和凝胶进行免疫沉淀以研究蛋白质-蛋白质相互作用

The scope of our research encompasses exploring the molecular mechanisms that regulate the development and function of brown and beige fat. Trunks, friction factors and co-factors such as the PRDM 16 complex, NFIA, and PPR alpha ELK-1 complex that we have more recently found control brown and beige fat cell fate and function. In addition, immune cell adipocyte crosstalk have been identified as key players.To advance research in your field, we employ several cutting edge technologies. These include quantitative PCR and chromatin immunoprecipitation for analyzing changes in gene expression as well as call immunoprecipitation and mass spectrometry to investigate alterations in protein-protein interactions, PPIs, associated with differentiation into beige adipocytes. Despite following the standard protocol for immunoprecipitation, detecting PPIs often proves challenging.Protein degradation is the primary cause of this issue, which we must carefully avoid. This protocol is simpler and less expensive than other techniques for examining PPIs, and does not include freeze thawing in the process, thus reducing protein degradation as much as possible.

洗脱表位标签沉淀蛋白和免疫印迹法用于蛋白质-蛋白质相互作用研究

将表位标记的蛋白质样品在 5， 000G 和 4 摄氏度下离心 30 秒。在冰上等待一分钟，让珠子变得平坦。然后丢弃上清液。将 1 毫升含有蛋白酶抑制剂的蛋白裂解缓冲液加入试管中，混合内容物。在 30 摄氏度的温度下将管子旋转约 30 秒循环 5 分钟。再次离心，将试管置于冰上一分钟，使珠子变平，然后丢弃上清液。现在，加入 10 微升样品缓冲液，将样品在 98 摄氏度下煮沸 10 分钟。将样品在 5， 000G 下在 4 摄氏度下离心 30 秒。将色谱柱放入蛋白质吸收低的新管中，并使用带有切口吸头的移液管将凝胶转移到色谱柱中。在 4 摄氏度下以 9、730G 离心一分钟并收集流出物。将凝胶放入电泳室中，并在凝胶周围加入tris-甘氨酸-SDS缓冲液至适当体积。将洗脱的蛋白质样品加载到十二烷基硫酸钠聚丙烯酰胺凝胶上。在 100 伏和 400 毫安下进行电泳。将凝胶转移到聚偏二氟乙烯膜上，并在室温下用 5% 脱脂牛奶吸干膜印迹一小时。在振荡器上用PBS聚氧乙烯脱水山梨糖醇单月桂酸酯洗涤膜三次，以最高速度洗涤五分钟。最后，将膜与一抗一起在 4 摄氏度的振荡器上孵育过夜。在转染的 HEK 293 细胞中，免疫印迹证实了转染 DYKDDDDK-PRDM16 和 HA-EHMT1 的组中 DYKDDDDK-PRDM16 和 HA-EHMT1 之间的关联。

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Research

JoVE Journal

Biology

Eluting Epitope Tag Precipitated Proteins and Immunoblotting for Protein-Protein Interaction Studies

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Video: Eluting Epitope Tag Precipitated Proteins and Immunoblotting for Protein-Protein Interaction Studies

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and transcriptional regulation. Therefore, understanding PPIs is an important starting point for further investigation of the function of the target protein. In this study, we propose a simple method to determine the binding of two target proteins by introducing mammalian expression vectors into HEK-293 cells using the polyethylenimine method, lysing the cells in homemade protein lysis buffer, and pulling down the target proteins on an epitope tag affinity gel. In addition, the PPI between the various epitope tag fused proteins can be confirmed by using affinity antibodies against each tag instead of the epitope tag affinity gel. This protocol could also be used to verify various PPIs, including nuclear extracts, from other cell lines. Therefore, it can be used as a basic method in a variety of PPI experiments. Proteins degrade by extended time course and repeated freeze-thaw cycles. Therefore, cell lysis, immunoprecipitation, and immunoblotting should be performed as seamlessly as possible.

Protein-protein interactions are important for elucidating the function of target proteins, and co-immunoprecipitation (co-IP) can easily confirm PPIs. We transiently transfected a plasmid encoding an epitope-tagged protein into HEK-293 cells and developed an immunoprecipitation method to easily confirm the binding of two target proteins.

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and ...