金年会 | JINNIAN.COM

The state of eukaryotic cells is influenced by both endogenous and exogenous factors, and the end point of all signaling pathways is DNA. DNA constitutes chromatin through nuclear protein complexes, an important site of action for chromatin gene regulation. Studies of transcriptional activators and co-inhibitors have revealed a novel regulatory mechanism called "histone codes" in which information is present in post-transcriptional modifications of histones. Such modifications include histone phosphorylation, acetylation, methylation, ADP-ribosylation and the like. As more and more histone core structural regions and carboxy-terminal modifications are identified, the role of histone codes in controlling and regulating gene function is becoming increasingly clear. The enzymes involved in the modification are classified according to their effects: for example, histidine acetyltransferase (HATs) can transfer acetyl groups to histones; histone deacetylases (HDACs) can remove acetyl groups on amino acids; Histone methyltransferases (HMTs) can transfer methyl groups to histones. Different histidine-modified markers correspond to different biological processes, which can serve as regulatory sites for regulatory factors and can also be used to alter chromatin structure.


Gel electrophoresis mobility shift assay (EMSA) is currently the common method for studying the binding of transcriptional regulatory proteins to their corresponding nucleotide sequences, but the results obtained by this in vitro assay are due to the fact that many transcriptional regulatory proteins have similar or identical DNA binding sites. It does not necessarily reflect the binding status of transcriptional regulatory proteins and DNA in vivo. Chromatin immunoprecipitation assay (ChiP) is a method developed based on in vivo analysis that truly and completely reflects regulatory proteins that bind to DNA sequences. It is currently the best way to identify genomic regions that bind to a particular protein or to identify proteins that bind to a particular genomic region. The combination of ChiP technology and chip technology facilitates the determination of chromosomal protein distribution patterns and histone modifications across the genome.

Figure: Schematic diagram of ChiP technology:

Formaldehyde treatment crosslinks proteins with DNA;

Ultrasound breaks chromatin into a certain size;

Precipitating a protein-DNA cross-linking complex by an antibody;

Release cross-linking and purify DNA;

Real-time quantitative PCR to detect the amount of DNA


application:

Antibodies to histone modification enzymes as "biomarkers" for transcriptional regulation analysis

Drug development research

Mitosis research

DNA loss and apoptosis analysis


Kit components 

ssDNA/Protein A Agarose

ssDNA/Protein A agarose 

ssDNA/Protein G Agarose

ChIP Dilution Buffer

Low salt wash buffer

High salt wash buffer

LiCl Wash Buffer

TE Buffer

0.5M EDTA

5M NaCL

1M Tris-HCl, pH 6.5

SDS Lysis Buffer

Please download the file for more content...