Protein-Protein Interaction Analysis Service
Alpha Lifetech can also provide customers with protein assays, protein interaction analysis, protein-protein interaction assay (CO-IP, Western Blot, Chromatin immunoprecipitation assay), and protein function assay to meet the experimental needs of different customers.
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Protein-Protein Interaction Analysis Service
Alpha Lifetech has been deeply engaged in protein assays for many years built a sound platform for protein function assay, and mastered a variety of technical means to verify protein interaction, which can save scientific research or project research time while delivering stable results.
Alpha Lifetech can also provide customers with protein assays, protein interaction analysis, protein-protein interaction assay (CO-IP, Western Blot, Chromatin immunoprecipitation assay), and protein function assay to meet the experimental needs of different customers.
Introduction To Protein-Protein Interaction Assay
Proteins are the most critical executive molecules in any form of life responding to the instructions stored in genetic materials. More frequently, proteins do their jobs by acting as role players that interact with other proteins, which is more evident when the function of a protein is examined in the real context of a cell. Identifying the interactions between proteins is very crucial for the biochemistry investigation of an individual protein. Protein interaction analysis of common kinds as follows:
Co-immunoprecipitation Assay
The principle of the co-immunoprecipitation method is to mix the target protein, specific antibody, and protein A/G magnetic beads for incubation, the supernatant of the unbound protein is discarded through the adsorption of the magnetic beads on the magnetic rack, and the magnetic beads are washed to remove the protein and other impurities unless specifically bound.
Fig.1 Schematic diagram of Co-IP assays. (Reference source: Coimmunoprecipitation assay - PubMed (nih.gov))
Pull-down Assay
The pull-down assay is to fix the known protein on the magnetic bead and add the substance to be detected. The eluent is detected for the adsorption of interacting proteins.
In 1988, Smith et al. purified GST fusion protein, which was applied in pull-down assays and is called Gst pull down that is to add a GST tag to the target protein, capture the interacting protein through GSH, eluate the binding, and verify it through WB assays.
Fig.2 Schematic diagram of GST pull-down assays. (Reference source: GST Pull-Down Assay to Study PIF4 Binding In Vitro - PubMed (nih.gov) )
His pull-down is to use metal ions such as nickel or cobalt as the medium, purify the protein through affinity chromatography, and eluate the binding through WB experiment for verification.
In addition, there are DNA Pull-down (Protein dna binding assay), RNA Pull-down (Protein rna interaction assay), and small molecule pull down assays.
DNA Pull-down (Protein dna binding assay) is an in vitro method of determining the DNA binding of protein. Whether specific proteins bind to target DNA was detected by WB assays; Unknown DNA fragments bound to proteins, which DNA fragments are known by MS detection
RNA Pull-down (Protein rna interaction assay) is an in vitro method for determining RNA binding to proteins. Whether specific proteins bind to target RNA was detected by WB assays; Unknown RNA fragments bound to proteins, which RNA fragments are known by MS detection
SM pull-down (small molecule pull-down assays) can find target proteins that bind specifically to small molecules, and when combined with MS, small molecule target proteins can be accurately screened, which can be divided into the in vitro labeling method and biological orthogonal method.
WB Experiment
WB experiment (also known as Western Blot experiment or Western blot), the essence is the specific reaction of antigen and antibody, the basic principle is to separate denatured proteins through SDS polyacrylamide gel electrophoresis and transfer it to a solid phase carrier (such as PVDF membrane, NC membrane) via membrane transfer (wet or semi-dry rotation). Following blocking (using BSA, buttermilk, etc.), the primary antibody is applied to bind specifically to the protein, and the fluorescein-labeled secondary antibody is added after washing the film. by binding the second antibody with the primary antibody, the target protein can be observed by substrate color development and chemiluminescence. It is generally used to determine whether a specific protein is expressed in the sample and roughly analyze its expression level.
Protein-Protein Interaction Method comparison
The protein pull-down assays are similar to the Co-IP assays, both of which belong to the detection of protein interaction experiments. The difference between the two methods is that the Co-IP assays obtain the interacting proteins of known proteins, which has a certain authenticity but can‘t confirm whether the interacting proteins interact directly or indirectly. Pull-down assays are to find the unknown protein interacting with the known protein. It can directly confirm whether the known protein interacts with the detected protein but can’t know the binding situation in vivo.
Co-immunoprecipitation technology is used to analyze whether there is an interaction between two or more proteins, based on immunoprecipitation experiments, Compared with other protein interaction analysis of experimental techniques (GST Pull-down, Western Blot, Chromatin immunoprecipitation assay, etc.), Co-IP technology has higher specificity, sensitivity, and high repeatability, which can avoid the interference of non-specific binding and reflect the interaction of proteins in the natural state.
| GST Pull down | Co-IP | WB | |
|---|---|---|---|
| Advantage | *Easy to operate *Suitable for the purification of various proteins
|
*For in vivo interaction analysis
*It can be identified with downstream protein
|
*Strong specificity *Qualitative and quantitative in vitro |
| Limitation |
*Maybe nonspecific binding
*Further validation of the interaction is required
|
*Strong dependence on antibodies *Nonspecific binding may occur
|
*Time-consuming
*Difficult to use for large-scale protein analysis
|
| Application Example |
*Identify interactions
*Purified protein complex
|
*Study signal transduction
*Disease mechanism
|
Subsequent analysis of protein-protein, protein-DNA, and protein-RNA interactions |
Customized
Customize services to meet your needs
Experience
Years of protein production experience
Technology
High quality delivery results
Experiment Process
Strict quality management process
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2018-07-16
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