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

Analys av proteininteraktion i HEK-293-celler med hjälp av co-immunoprecipitation

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.

Författare i fokus: Att reda ut de molekylära mekanismerna för reglering av bruna och beige adipocyter

Immunoprecipitering med en anti-epitoptagg-affinitetsgel för att studera protein-proteininteraktioner

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 av transfekterat HEK293-protein med Protein G Gel och infångning av proteinkomplex med hjälp av Epitope Tag Affinity Gel

För att börja, tillsätt 40 mikroliter av en till en protein G-gelslurry i röret som innehåller proteinprovet. Rotera provet i en timme på en rotator i cirka 30 sekunders cykler vid fyra grader Celsius. Centrifugera sedan provet vid 12 000 G i 20 sekunder vid fyra grader Celsius.Lägg röret på is i en minut så att pärlorna kan plana ut. Samla upp supernatanten och överför den till ett nytt rör på 1,5 milliliter med låg proteinabsorption. Ta bort alikvoten för inmatning i ett separat 1,5 milliliters rör.Tillsätt fyra gånger den koncentrerade provbufferten till tillförseln för att göra en lika stor utspädning och värm vid 98 grader Celsius i 10 minuter. Tillsätt sedan 20 till 30 mikroliter av en en-till-en-epitoptaggslurry till provet. Rotera provet i två timmar på en rotator i cirka 30-sekunders cykler vid fyra grader 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 ...

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

Celllys av transfekterade HEK293-celler och proteinprovberedning för immunprecipitering

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

Eluering av epitoptagg utfällda proteiner och immunoblotting för studier av protein-proteininteraktion