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

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.

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

Centrifugue a amostra de proteína marcada com epítopo a 5.000 G por 30 segundos a 4 graus Celsius. Espere um minuto no gelo para permitir que as contas se nivelem. Em seguida, descarte o sobrenadante.Adicione um mililitro de tampão de lise de proteína contendo inibidor de protease no tubo e misture o conteúdo. Gire o tubo em um rotador por ciclos de aproximadamente 30 segundos a 4 graus Celsius por cinco minutos. Centrifugue novamente e coloque o tubo no gelo por um minuto para nivelar as contas antes de descartar o sobrenadante.Agora, adicione 10 microlitros de tampão de amostra e ferva a amostra a 98 graus Celsius por 10 minutos. Centrifugue a amostra a 5.000 G por 30 segundos a 4 graus Celsius. Coloque uma coluna em um novo tubo com baixa absorção de proteínas e transfira o gel para uma coluna usando uma pipeta com ponta cortada.Centrifugue a 9.730G por um minuto a 4 graus Celsius e colete o fluxo. Coloque o gel na câmara de eletroforese e adicione tampão tris-glicina-SDS até o volume apropriado ao redor do gel. Carregue as amostras de proteína eluída em um gel de poliacrilamida de dodecil sulfato de sódio.Realize eletroforese a 100 volts e 400 miliamperes. Transfira o gel para uma membrana de fluoreto de polivinilideno e seque a membrana com 5% de leite desnatado por uma hora em temperatura ambiente. Lave a membrana três vezes com monolaurato de polioxietileno sorbitano PBS em um agitador em velocidade máxima por cinco minutos.Finalmente, incube a membrana com o anticorpo primário durante a noite em um agitador a 4 graus Celsius. Em células HEK 293 transfectadas, o immunoblotting confirmou a associação entre DYKDDDDK-PRDM16 e HA-EHMT1 nos grupos transfectados com DYKDDDDK-PRDM16 e HA-EHMT1.

Research

JoVE Journal

Biology

Protein Interaction Analysis in HEK-293 Cells Using Co-Immunoprecipitation

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

Author Spotlight: Unraveling the Molecular Mechanisms of Brown and Beige Adipocyte Regulation

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

Biologia

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

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