{"id":779,"date":"2020-03-25T08:18:51","date_gmt":"2020-03-25T13:18:51","guid":{"rendered":"http:\/\/www.creative-diagnostics.com\/blog\/?p=779"},"modified":"2020-12-09T01:49:31","modified_gmt":"2020-12-09T06:49:31","slug":"talking-about-protein-interaction-detection-method-ii","status":"publish","type":"post","link":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/talking-about-protein-interaction-detection-method-ii\/","title":{"rendered":"Talking About Protein Interaction Detection Method II"},"content":{"rendered":"

Proteins are essential participants in biological processes and they are responsible for initiating and performing tasks in biological systems. From the duplication of genetic material to cellular senescence and death, every biological process in an organism requires functional coordination between several and even hundreds of proteins. Changes in protein function and structure often disrupt this balance of protein-protein interactions cause disease. To further investigate protein interactions, scientists have developed a variety of inspection methods. The main methods currently used to study protein interactions include two-dimensional electrophoresis, phage display technology, GST pull-down, yeast two-hybrid, tandem affinity purification, bimolecular fluorescence complementary technology, protein chips, Far Western blotting and co-immunoprecipitation.<\/p>\n

\"\"<\/a>
Figure 1. Schematic diagram of membrane protein interactions.<\/figcaption><\/figure>\n

Tandem Affinity Purification<\/a><\/strong>
\nTandem affinity purification (TAP \/ MS) is to construct a vector containing fusion expression of the target gene and two different epitope tags (such as Flag, HA, His, CBP, SBP, etc.).\u00a0After transfecting the cells with the vector and expressing the bait protein with two tags, the cells were lysed and the total protein was extracted.\u00a0After purification and elution with two different tag resins, the false-positive proteins were removed to the greatest extent.\u00a0Finally, the eluate was placed in a mass spectrometer and analyzed to identify proteins that interacted with the bait protein.<\/p>\n

Bimolecular Fluorescence Complementary Technology<\/strong>
\nThe technology is to cut the fluorescent protein at some specific sites to form two non-fluorescent N- and C-terminal polypeptides, called N fragments and C fragments.\u00a0When these two fragments are co-expressed in cells or mixed in vitro, they cannot spontaneously assemble into a complete fluorescent protein, so they cannot produce fluorescence when excited by excitation light.\u00a0However, when the two fluorescent protein fragments are respectively linked to a group of interacting target proteins, and the two fusion proteins are co-expressed in the cell or mixed in vitro.\u00a0And then, the two fluorescent protein fragments are reconstructed by the interaction of the target protein, and emit fluorescence under the excitation of the fluorescent protein’s excitation light.\u00a0Finally, the results can be judged by comparing the fluorescence intensity and density.<\/p>\n

Protein Chip<\/strong>
\nThe research object of protein chip technology is protein.\u00a0The principle is to carry out special chemical treatment on the solid phase carrier, and then fix the known protein molecular products on it (such as enzymes, antigens, antibodies, receptors, ligands, cytokines, etc.).\u00a0According to the characteristics of these biomolecules, capture the test protein that can specifically bind to it (exist in serum, plasma, lymph, interstitial fluid, urine, exudate, cell lysate, secretion, etc.).\u00a0After washing and purification, the target protein is obtained.\u00a0Finally, confirm and biochemically analyze the target protein.<\/p>\n

Far Western Blot<\/strong>
\nFar Western Blot is another method for analyzing protein interactions.\u00a0It requires the detection of proteins separated by gel electrophoresis together with the labeled bait protein, and then other methods to determine the protein of this interaction.\u00a0In recent years, far western blot analysis has been used to identify receptor ligand interactions and screening libraries for interacting proteins.\u00a0Using this method of analysis, the effects of post-translational modifications on protein interactions can be studied.\u00a0Using synthetic peptides as probes to detect interacting sequences, protein-protein interactions can be identified without the use of antigen-specific antibodies.<\/p>\n

Co-immunoprecipitation<\/a><\/strong>
\nCo-immunoprecipitation is an effective method for determining the physiological interaction of two proteins in intact cells based on the specific interaction between antibodies and antigens.\u00a0When cells are lysed under non-denaturing conditions, many protein-protein interactions present in intact cells are preserved.\u00a0When protein A is immunoprecipitated with antibodies that are pre-cured on argarose beads, protein B bound to protein A in the body can also be precipitated together.\u00a0Then the protein B was detected through denaturation and separation to prove the interaction between the two.<\/p>\n

The above is the main method of protein interaction. If you need efficient and timely testing services, please visit our main website to learn more.<\/p>\n","protected":false},"excerpt":{"rendered":"

Proteins are essential participants in biological processes and they are responsible for initiating and performing tasks in biological systems. From the duplication of genetic material to cellular senescence and death, every biological process in an organism requires functional coordination between several and even hundreds of proteins. Changes in protein function and structure often disrupt this […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":[],"categories":[59],"tags":[64],"aioseo_notices":[],"_links":{"self":[{"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/posts\/779"}],"collection":[{"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/comments?post=779"}],"version-history":[{"count":6,"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/posts\/779\/revisions"}],"predecessor-version":[{"id":799,"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/posts\/779\/revisions\/799"}],"wp:attachment":[{"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/media?parent=779"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/categories?post=779"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.creative-diagnostics.com\/blog\/index.php\/wp-json\/wp\/v2\/tags?post=779"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}