什么是 ChIP，为什么有用？

ChIP immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome for a specific protein of interest, giving invaluable insights into the regulation of gene expression. ChIP involves the selective enrichment of a chromatin fraction containing a specific antigen. Antibodies that recognize a specific protein or protein modification are used to determine the relative abundance of that antigen at a specific locus or loci. ChIP can be used to compare enrichment of proteins, map protein modifications, or quantify a protein modification during a time course. ChIP is frequently used in studies that focus on cellular differentiation, tumor suppressor gene silencing, the effect of histone modifications on gene expression, and other studies involving transcriptional activation and repression.

ChIP 的工作原理

Your checklist: the most important parts of your ChIP assay

• Optimize crosslinking

  Crosslinking: Fresh formaldehyde is used to form the reversible DNA-protein crosslinks before immunoprecipitation. Formaldehyde is usually not effective to crosslink indirectly-bound proteins.

  
  

  Your solution for higher order or dynamic protein interactions for efficient protein-protein stabilization is Diagenode ChIP Crosslink Gold.

  • Use fresh and high quality formaldehyde without precipitates
  • Optimize the crosslinking time. Avoid excessive crosslinking time as binding efficiency is decreased
  • Formaldehyde is used to form the reversible DNA-protein crosslinks before immunoprecipitation but is usually not effective to crosslink indirectly-bound proteins. Your solution for higher order or dynamic protein interactions for efficent protein-protein stabilization is Diagenode ChIP Crosslink Gold.
• Optimize chromatin shearing

  Shearing: You need fragments that are optimally-sized for effective immunoprecipitations and accurate downstream analysis sush as library prep.

  
  

  Solutions
  

  Bioruptor Pico for controlled shearing to achieve the right size range you need for ChIP
  Chromatin Shearing Optimization Kits

  Fragment size requirements

  Fragments between 100-500 bp are suitable for the majority of ChIP experiments. Many ChIP-seq experiments are commonly performed with chromatin in the 100-800 bp range. Generally, a tighter fragment distribution is recommended for ChIP-seq than for ChIP-qPCR. Typically, 100-300 bp is compatible with histone ChIP-seq while a larger range is more suitable for transcriptional factors, indirectly-bound proteins, and distant interacting proteins.

  Achieving best fragmentation

  Use the Bioruptor Pico: More than 8,000 peer-reviewed scientific papers cite the Bioruptor. The range of chromatin size can be easily fine-tuned with the Bioruptor Pico. The Bioruptor Pico delivers high performance ultrasound in with the needed temperature control, allowing optimal shearing efficency. Remember to use the appropriate tubes (available from Diagenode).

  Use the Chromatin Shearing Optimization Kits: It is important to establish optimal conditions to shear crosslinked chromatin (protein-DNA). Usually this process requires optimizing SDS concentration, which is laborious. With the Shearing Optimization Kit, optimization is fast and easy - we provide optimization reagents with varying concentrations of SDS, optimized for both transcription factors and histones.

  Read the full Chromatin Shearing Guide: you'll know everything and more about best shearing strategy.

• Immunoprecipitation: use validated antibodies

  Successful ChIP-seq and ChIP-PCR is critically dependent on the quality of ChIP antibodies

  Always choose antibodies validated in ChIP. Antibodies that perform well in other applications may not perform well in ChIP. Diagenode specializes in highly-qualified antibodies with the most rigorous QC and validation procedures (target specificity, characterization, and performance in ChIP).

  Make sure validation data is available from the supplier. Diagenode publishes all data in the datasheets right here on our website.

  Either monoclonals or polyclonals work for ChIP. Monoclonals exhibit less nonspecific binding and have lower variability but may not be as effective for isolating rare epitopes. Polyclonals recognize multiple epitopes, theoretically increasing the chance of nonspecific binding. However, this quality can be controlled depending on manufacturer's practices. At Diagenode, we test every lot and assure low batch-to-batch variability.

• Immunoprecipitation: use validated and complete ChIP kits

  The optimal kits for ChIP include all the reagents needed for ChIP-qPCR or ChIP-seq. Diagenode's kits contain everything you need for start-to-finish ChIP including primers to check the quality of the ChIP by qPCR as well as positive and negative controls.

  Make life easier. Choose one of our complete and validated kits that includes all immunoprecipitation and wash buffers, negative and positive control antibodies, and control primers. All Diagenode kits are complete and validated.

  Run a negative control. To control for specific of your ChIP antibody, run a negative control such as IgG (included in Diagenode kits).

  Run a positive control. We include the antibody to abundant histone H3K4me3 for any required ChIP troubleshooting. Our transcription factor kits have the added benefit of including CTCF antibody for the highly prevalent transcription factor.

  Use the ChIP-seq grade primers to check enrichment by qPCR before proceeding to further analysis (primers are included in all of our kits).

  Decide what kit is right for you by clicking here

• Consider automating for easy, reproducible ChIP

  It is easy to accomplish complete walk-away ChIP and ChIP library preparation with the IP-Star Automation System. Designed to process up to 16 samples in parallel from start to finish, the automation system uses magnetic bead technology and automated dispensing for maximum reproducibility of your results.

  Read more

• Consider services for any part of your ChIP assay

  You can have our epigenetics sample prep experts process any part of your ChIP assay for you from immunoprecipitation to library preparation for ChIP-seq to full sequencing, analysis, and bioinformatics.

  Read more

ChIP applications

Locate the genome-wide binding sites of transcription factors and histones

Uncover DNA sequences bound by specific proteins

Understand DNA-protein interactions in disease

Uncover protein modifications that influence gene expression and transcription and chromatin structure

What are some good resources for learning more about ChIP and ChIP solutions from Diagenode?