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

HEK-293 Hücrelerinde Ko-İmmünopresipitasyon Kullanılarak Protein Etkileşim Analizi

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.

Yazar Spotlight: Kahverengi ve Bej Adiposit Regülasyonunun Moleküler Mekanizmalarının Çözülmesi

Protein-Protein Etkileşimlerini İncelemek için Anti-Epitop Etiket Afinite Jeli ile İmmünopresipitasyon

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.

Protein-Protein Etkileşim Çalışmaları için Epitop Etiketi Çökeltilmiş Proteinlerin Ayrıştırılması ve İmmünoblotlama

Epitop etiketli protein örneğini 5.000G'de 4 santigrat derecede 30 saniye santrifüjleyin. Boncukların düzleşmesine izin vermek için buz üzerinde bir dakika bekleyin. Ardından süpernatanı atın.Tüpe proteaz inhibitörü içeren bir mililitre protein lizis tamponu ekleyin ve içeriği karıştırın. Tüpü bir döndürücü üzerinde yaklaşık 30 saniyelik döngüler için beş dakika boyunca 4 santigrat derecede döndürün. Tekrar santrifüjleyin ve süpernatanı atmadan önce boncukları düzleştirmek için tüpü bir dakika boyunca buzun üzerine yerleştirin.Şimdi 10 mikrolitre numune tamponu ekleyin ve numuneyi 98 santigrat derecede 10 dakika kaynatın. Numuneyi 5.000G'de 4 santigrat derecede 30 saniye santrifüjleyin. Düşük protein emilimine sahip yeni bir tüpe bir sütun yerleştirin ve jeli kesilmiş uçlu bir pipet kullanarak bir sütuna aktarın.9, 730G'de 4 santigrat derecede bir dakika santrifüjleyin ve akışı toplayın. Jeli elektroforez odasına yerleştirin ve jelin etrafındaki uygun hacme kadar tris-glisin-SDS tamponu ekleyin. Ayrıştırılmış protein örneklerini bir sodyum dodesil sülfat poliakrilamid jel üzerine yükleyin.100 volt ve 400 miliamperde elektroforez gerçekleştirin. Jeli bir poliviniliden florür membranına aktarın ve membran lekesini oda sıcaklığında bir saat boyunca %5 yağsız sütle kurulayın. Membranı PBS polioksietilen sorbitan monolaurat ile bir çalkalayıcı üzerinde beş dakika boyunca en yüksek hızda üç kez yıkayın.Son olarak, zarı birincil antikor ile gece boyunca 4 santigrat derecede bir çalkalayıcı üzerinde inkübe edin. Transfekte edilmiş HEK 293 hücrelerinde, immün bloklama, DYKDDDDK-PRDM16 ve HA-EHMT1 ile transfekte edilen gruplarda DYKDDDDK-PRDM16 ve HA-EHMT1 arasındaki ilişkiyi doğruladı.

eluting-epitope-tag-precipitated-proteins-immunoblotting-for-protein

Research

JoVE Journal

Biology

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

49 Görüntüleme.  Hiroshima University. Epitop etiketli protein örneğini 5.000G'de 4 santigrat derecede 30 saniye santrifüjleyin. Boncukların düzleşmesine izin vermek için buz üzerinde bir dakika bekleyin. Ardından süpernatanı atın.Tüpe proteaz inhibitörü içeren bir mililitre protein lizis tamponu ekleyin ve içeriği karıştırın. Tüpü bir döndürücü üzerinde yaklaşık 30 saniyelik döngüler için beş dakika boyunca 4 santigrat derecede döndürün. Tekrar santrifüjleyin ve süpernatanı atmadan ...

Video: Protein-Protein Etkileşim Çalışmaları için Epitop Etiketi Çökeltilmiş Proteinlerin Ayrıştırılması ve İmmünoblotlama

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 ...