preclearing of transfected hek293 protein with the protein g gel and capturing protein complexes using epitope tag affinity gel

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

使用 Protein G 凝胶预清除转染的 HEK293 蛋白，并使用 Epitope Tag Affinity Gel 捕获蛋白质复合物

首先，将 40 微升一比一蛋白质 G 凝胶浆液加入含有蛋白质样品的试管中。在旋转器上以大约 30 秒的周期在 4 摄氏度下旋转样品一小时。接下来，将样品以 12， 000 G 的速度在 4 摄氏度下离心 20 秒。将试管放在冰上一分钟，让珠子趋平。收集上清液并将其转移到低蛋白吸收的新 1.5 毫升管中。取出等分试样，输入到单独的 1.5 毫升管中。向输入中加入 4 倍的浓缩样品缓冲液以进行等稀释，并在 98 摄氏度下加热 10 分钟。接下来，向样品中加入 20 至 30 微升的一对一表位标签浆液。在旋转器上以大约 30 秒的周期在 4 摄氏度下旋转样品两小时。

preclearing-transfected-hek293-protein-with-protein-g-gel-capturing

Research

JoVE Journal

Biology

Preclearing of Transfected HEK293 Protein with the Protein G Gel and Capturing Protein Complexes Using Epitope Tag Affinity Gel

45 次观看. 首先，将 40 微升一比一蛋白质 G 凝胶浆液加入含有蛋白质样品的试管中。在旋转器上以大约 30 秒的周期在 4 摄氏度下旋转样品一小时。接下来，将样品以 12， 000 G 的速度在 4 摄氏度下离心 20 秒。将试管放在冰上一分钟，让珠子趋平。收集上清液并将其转移到低蛋白吸收的新 1.5 毫升管中。取出等分试样，输入到单独的 1.5 毫升管中。向输入中加入 4 倍的浓缩样品?...

视频: 使用 Protein G 凝胶预清除转染的 HEK293 蛋白，并使用 Epitope Tag Affinity Gel 捕获蛋白质复合物

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 ...