Chromatin Immunoprecipitation Assay (ChIP)

Chromatin immunoprecipitation assay is an antibody-based technology used to investigate the interaction between proteins and DNA in the cell. Chip assays monitor transcriptional regulation and identify linkages between the genome and proteome through histone modifications (epigenetics) or transcription factor-DNA binding interactions.

Cross-link DNA and proteins in cells under physiological conditions cut chromatin into small fragments by sonication or enzymatic treatment. Using the specific recognition reaction of antigen and antibody, the DNA fragments bound to the target protein are precipitated to enrich the DNA fragments with histone modification or transcriptional regulation. The DNA sequence of this enriched fragment is then detected by a variety of downstream detection techniques (quantitative PCR, gene chip, sequencing, etc.).

• Crosslinking
  Crosslink DNA and associated proteins on chromatin in living cells or tissues with formaldehyde or other crosslinking agents.
• Cell lysis
  Dissolve cell membranes by a detergent solution to release cellular components and then bring protein-DNA complexes into solution.
• Chromatin preparation (shearing/digestion)
  Shear the DNA-protein complexes (chromatin-protein) into ~500 bp DNA fragments by sonication or nuclease digestion.
• Immunoprecipitation
  Bind the antibody against the protein of interest to the protein-DNA complex, and then precipitate the complex.
• Decrosslinking and DNA purification
  Broke the cross-links between the protein and the DNA by heat incubation or by protease digestion.
• DNA analysis
  Analyze the degree of enrichment of DNA via PCR, qPCR, microarray, or NGS.

Fig. 1 The basic flow chart for ChIP.

• Cross-linked ChIP (XChIP)
  X-ChIP can be used with histone and non-histone proteins. It generally requires less cellular starting material than N-ChIP and minimizes the chances of chromatin protein loss during extraction to detect transient protein interactions. However, the precipitation step is inefficient and downstream analysis requires DNA amplification by PCR.
• Native ChIP (NChIP)
  In N-ChIP, no fixing agent is used to crosslink proteins to the chromatin. Antibodies can be recognized and bound to their target antigens more effectively. However, it is only capable of detecting histones. In addition, protein binding may be lost during the chromatin digestion and immunoprecipitation steps, which may bias data or impede proper analyses.

• Screen and identify protein binding sites on the whole chromosome efficiently and accurately in combination with NGS.
• Can be coupled to many commonly used molecular biology techniques.

• For the analysis of proteins interacting within a native chromatin environment.
• Determine the allele-specific transcription factor binding patterns and measure long-range enhancer binding of specific proteins.
• Uncover an extraordinarily rich and dynamic chromatin environment.
• Provide useful end-point measurement for delineation of components within the signal transduction pathways

Laboratory Incubators

An incubator is a device used to grow and maintain microbiological cultures or cell cultures. It is made up of a chamber with a regulated temperature. An incubator maintains optimal temperature, humidity, and other conditions such as the CO2 and oxygen content of the atmosphere inside. Incubators are essential for much experimental work in cell biology, microbiology, and molecular biology.

Laboratory Centrifuges

A centrifuge is any device that applies a sustained centrifugal force—that is, a force due to rotation. The widest use of centrifuges is for the concentration and purification of materials in suspension or dissolved in fluids. Suspended particles denser than the suspending liquid tend to migrate toward the periphery, while those less dense move toward the centre.

Pipette

Pipette is a small piece of apparatus which typically consists of a narrow tube into which fluid is drawn by suction (as for dispensing or measurement) and retained by closing the upper end. Pipettes are a common laboratory tool and come in a multitude of channels, displacements and construction materials. There are different pipettes for all kinds of uses within the lab.

Laboratory Refrigeration

Laboratory refrigerators are designed with special features and options for use in the laboratory, including a range of refrigerator and freezer accessories. The primary function of a laboratory refrigerator is to maintain a defined, internal storage temperature in order to provide secure storage and protection of temperature-sensitive products, samples, specimens, chemicals, drugs, solutions, and other substances.

Electrophoresis Systems

Electrophoresis equipment applies an electric charge to molecules, causing them to migrate towards their oppositely charged electrode. The technique is found in all research and clinical laboratories utilizing DNA and protein applications, and is divided into gel and capillary techniques. Choice of electrophoresis equipment depends on the molecule of study, appropriate method of separation and downstream application.

Homogenizers / Sonicators

Homogenizers, commonly referred to as "sonicators," disrupt tissues and cells through cavitation and ultrasonic waves. Basically, a homogenizer has a tip which very rapidly vibrates, causing bubbles in the surrounding solution to rapidly form and collapse. This creates shear and shock waves which tear apart cells and particles. Homogenizers / Sonicators are great for breaking apart cells and subcellular structures in suspension. They are not good for homogenizing intact tissue.

Vortexer

Vortexers, or vortex mixers, have very fast, small orbits (usually between 2 and 4 mm) and they are named for their ability to form a vortex in the liquid that they are mixing. are commonly used in laboratories to mix small liquid volumes for cell disruption or homogenization. Constructed of rugged, corrosion-resistant materials, the instruments will endure heavy everyday usage.

STEMart provides you with a variety of ChIP equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for ChIP, please feel free to contact us.

Reference

• Gade P, Kalvakolanu DV. Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity. Methods Mol Biol. 2012;809:85-104. doi: 10.1007/978-1-61779-376-9_6. PMID: 22113270; PMCID: PMC3891665.