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

Análise de interação proteica em células HEK-293 usando co-imunoprecipitação

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 em destaque: Desvendando os mecanismos moleculares da regulação dos adipócitos marrons e bege

Imunoprecipitação com um gel de afinidade anti-epítopo para estudar interações proteína-proteína

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.

Pré-limpeza da proteína HEK293 transfectada com o gel de proteína G e captura de complexos de proteínas usando gel de afinidade de etiqueta de epítopo

Para começar, adicione 40 microlitros de pasta de gel de proteína G um a um no tubo que contém a amostra de proteína. Gire a amostra por uma hora em um rotador em ciclos de aproximadamente 30 segundos a quatro graus Celsius. Em seguida, centrifugar a amostra a 12 000 G durante 20 segundos a quatro graus Celsius.Coloque o tubo no gelo por um minuto para permitir que as contas se nivelem. Recolha o sobrenadante e transfira-o para um novo tubo de 1,5 mililitros com baixa absorção de proteínas. Remova a alíquota para entrada em um tubo separado de 1,5 mililitro.Adicione quatro vezes o tampão de amostra concentrado à entrada para fazer uma diluição igual e aqueça a 98 graus Celsius por 10 minutos. Em seguida, adicione 20 a 30 microlitros de uma pasta de etiqueta de epítopo um para um à amostra. Gire a amostra por duas horas em um rotador em ciclos de aproximadamente 30 segundos a quatro graus Celsius.

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

43 visualizações.  Hiroshima University. Para começar, adicione 40 microlitros de pasta de gel de proteína G um a um no tubo que contém a amostra de proteína. Gire a amostra por uma hora em um rotador em ciclos de aproximadamente 30 segundos a quatro graus Celsius. Em seguida, centrifugar a amostra a 12 000 G durante 20 segundos a quatro graus Celsius.Coloque o tubo no gelo por um minuto para permitir que as contas se nivelem. Recolha o sobrenadante e transfira-o para um novo tubo de 1,5 mililitros com baixa absorção de...

Vídeo: Pré-limpeza da proteína HEK293 transfectada com o gel de proteína G e captura de complexos de proteínas usando gel de afinidade de etiqueta de epítopo

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 ...