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Creative Proteomics Scientists Share Precautions for Chromatin Immunoprecipitation Experiments

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Eukaryotic genomic DNA exists as chromatin. Therefore, studying the interaction between protein and DNA in chromatin environment is the basic way to elucidate the gene expression mechanism of eukaryotes.

— Eukaryotic genomic DNA exists as chromatin. Therefore, studying the interaction between protein and DNA in chromatin environment is the basic way to elucidate the gene expression mechanism of eukaryotes. Chromatin immunoprecipitation (CHIP) is currently the only method to study the interaction between DNA and proteins in vivo.

Its basic principle is to fix the protein-DNA complex in a living cell state and randomly cut it into small chromatin fragments within a certain length range, and then precipitate this complex by immunological methods to specifically enrich the target protein The bound DNA fragments are used to purify and detect the target fragments to obtain information about protein-DNA interactions.

CHIP can not only detect the dynamic effects of trans factors and DNA in vivo, but also can be used to study the relationship between various covalent modifications of histones and gene expression. In addition, the combination of CHIP and other methods has expanded its application scope. The CHIP-on-chip method established by combining CHIP and gene chips has been widely used for high-throughput screening of specific trans factor target genes; CHIP with in vivo footprinting methods is used to find the binding site of trans factor in vivo; RNA-CHIP is used to study the role of RNA in the regulation of gene expression. It can be seen that with the further improvement of CHIP, it will play an increasingly important role in the study of gene expression regulation.

Chromatin Immunoprecipitation (ChIP) is a method developed based on in vivo analysis, also known as binding site analysis. It has become the main method of epigenetic information research in the past decade. This technology helps researchers determine what histone modifications will occur at a particular location in the genome of the nucleus.

ChIP can not only detect the dynamic effects of trans factors and DNA in vivo, but also can be used to study the relationship between various covalent modifications of histones and gene expression. In recent years, this technology has been continuously developed and improved. Combining microarray technology to examine chromosomal activity in chromosomal gene expression regulatory regions is a very effective tool for in-depth analysis of major metabolic pathways for diseases such as cancer, cardiovascular disease, and central nervous system disorders.

Its principle is that while maintaining the combination of histone and DNA, through the use of a biological antibody corresponding to a specific histone tag, chromatin is cut into very small pieces and precipitated. IP is a method developed by utilizing the specific binding of an antigen protein and an antibody, and the phenomenon that "prorein A" of a bacterial protein specifically binds to an FC fragment of an immunoglobulin. At present, the multi-purpose refined prorein A is pre-bound and solidified on the beads of argarose, and after reacting with the solution and antibody containing the antigen, the prorein A on the beads can adsorb the antigen to achieve the purpose of purification.

The most important point of the experiment is the nature of the antibody. Different antibodies and antigen-binding capabilities are different, and dye-free binding may not be used in IP reactions. It is recommended to check the antibody manual carefully. In particular, the specificity of polyclonal antibodies is a problem.

Secondly, pay attention to the nature of the buffer in which the antigen is dissolved. Most antigens are cellular proteins, especially backbone proteins, which must be lysed by the buffer. For this reason, a buffer containing a strong surfactant must be used, although it may affect the binding of a part of the antigen-antibody.

On the other hand, if the cells are lysed with a weak surfactant, the cell proteins cannot be fully lysed. Even the dissolution also results in binding to other proteins, the epitope is blocked, affecting the binding to antibodies, and even if IP is successful, it is a tragic result of co-precipitation of many proteins and antibodies.

Third, in order to prevent protein degradation and modification, the antigen-dissolving buffer must be added to each inhibitor of the protein, and the experiment should be performed at low temperature. Before each experiment, first consider the antibody / buffer ratio. Too little antibody will not detect the antigen, too much will not settle on the beads, and the supernatant will remain. Too little buffer will not dissolve the antigen, too much will dilute the antigen.

Creative Proteomics can provide custom experimental design and refine Co-IP parameters based on customers’ needs. The company promises reliable Co-IP analysis of protein-protein interactions with its experienced scientists and technicians. Established in 2004, Creative Proteomics has gradually developed into an integrated company that provides proteomics, metabolomics, glycomics, and bioinformatics analysis services to researchers in the pharmaceutical, biotechnology, agriculture and nutrition industries, as well as academic and government organizations.

Contact Info:
Name: Creative Proteomics
Email: Send Email
Organization: Creative Proteomics
Address: 45-1 Ramsey Road, Shirley, NY 11967, USA,
Website: https://www.creative-proteomics.com/

Source URL: https://marketersmedia.com/creative-proteomics-scientists-share-precautions-for-chromatin-immunoprecipitation-experiments/88950072

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