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

Protein-Interaktionsanalyse in HEK-293-Zellen mittels Co-Immunpräzipitation

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.

Autor im Rampenlicht: Entschlüsselung der molekularen Mechanismen der Regulation von braunen und beigen Adipozyten

Immunpräzipitation mit einem Anti-Epitop-Tag-Affinitätsgel zur Untersuchung von Protein-Protein-Wechselwirkungen

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.

Preclearing des transfizierten HEK293-Proteins mit dem Protein-G-Gel und Einfangen von Proteinkomplexen mit Epitop-Tag-Affinitätsgel

Geben Sie zunächst 40 Mikroliter einer Eins-zu-Eins-Protein-G-Gel-Aufschlämmung in das Röhrchen mit der Proteinprobe. Drehen Sie die Probe eine Stunde lang auf einem Rotator in Zyklen von etwa 30 Sekunden bei vier Grad Celsius. Anschließend zentrifugieren Sie die Probe bei 12.000 g für 20 Sekunden bei vier Grad Celsius.Lege die Tube eine Minute lang auf Eis, damit sich die Perlen ausgleichen können. Sammeln Sie den Überstand und geben Sie ihn in ein neues 1,5-Milliliter-Röhrchen mit geringer Proteinabsorption. Entfernen Sie das Aliquot für die Eingabe in ein separates 1,5-Milliliter-Röhrchen.Geben Sie das Vierfache des konzentrierten Probenpuffers in den Eingang, um eine gleichmäßige Verdünnung zu erzielen, und erhitzen Sie ihn 10 Minuten lang auf 98 Grad Celsius. Geben Sie anschließend 20 bis 30 Mikroliter einer Eins-zu-Eins-Epitop-Tag-Aufschlämmung in die Probe. Drehen Sie die Probe zwei Stunden lang auf einem Rotator in etwa 30-Sekunden-Zyklen bei vier Grad Celsius.

Research

JoVE Journal

Biology

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

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 ...

Forschung

Biologie

Cell Lysis of Transfected HEK293 Cells and Protein Sample Preparation for Immunoprecipitation

Zelllyse von transfizierten HEK293-Zellen und Proteinprobenvorbereitung für die Immunpräzipitation

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

Elutierung von Epitop-Tag-präzipitierten Proteinen und Immunoblotting für Protein-Protein-Interaktionsstudien