Chromatin immunoprecipitation (ChIP) can be used as a tool to study protein:DNA complexes and identify protein-binding sites in the DNA. At BioLegend, we offer a wide-array of Go-ChIP-Grade™ antibodies and an enzymatic kit featuring a solid-state immunoprecipitation platform ideal for consistency between experiments.

Chromatin Immunoprecipitation is a powerful tool to study protein:DNA complexes and can be used to map transcription factor binding sites or to study epigenetic modifications. In this assay, chromatin bound proteins are crosslinked and the DNA is fragmented. Next, the sheared DNA:protein complexes are incubated with an antibody against the protein-of-interest and immunoprecipitated to enrich for bound DNA. Finally, the crosslinks are reversed and the DNA is eluted for analysis. Purified DNA can analyzed using qPCR, Next-Generation Sequencing, ChIP-on-chip analysis, etc.

Click on the steps below to learn more about how to optimize your ChIP assays.

 

 

Fixation and Chromatin Isolation
A successful ChIP experiment is dependent on high quality chromatin. Chromatin quality can be affected by lysis, fixation, and shearing conditions. As chromatin can degrade very quickly, it should be kept on ice throughout the experiment, and freeze/thaw cycles should be avoided.

Fixation
Formaldehyde is commonly used to reversibly cross-link proteins bound to DNA. For consistent crosslinking results, ensure that the fixation solution is made fresh and is methanol-free. For larger complexes or indirect protein interactions, formaldehyde can be used in combination with other cross-linkers such as EGS or DSG. However, other fixation methods, such as UV cross-linking are not reversible and are not suitable for ChIP applications.

Fixation conditions must be optimized for each experiment as over-fixing your samples may make them resistant to subsequent lysis and shearing steps. Additionally, over-fixation will make it more difficult to reverse cross-links and interfere with downstream analysis.

Cell lysis
Lysis buffers used for ChIP experiments contain detergents which can precipitate out of solution at cold temperatures. As such, lysis buffers should be warmed to room temperature to ensure precipitates are fully dissolved before use. Excess detergent in the lysis buffer may inhibit antibody binding during immunoprecipitation, so lysis buffer volume and composition must be optimized.

 

 

DNA Shearing
Chromatin can be sheared using two methods: sonication or enzymatic digestion. Regardless of the method chosen, chromatin should be sheared to fragments of 150-900 base pairs. Regardless of the method chosen, chromatin should be sheared to fragments of 100-900 base pairs. If the chromatin is over-sheared, it risks damaging the epitope bound by the protein or may prevent qPCR primers from recognizing the DNA fragments. If chromatin is under-sheared, it may cause an increase in non-specific binding.

Sonication
Sonication is a mechanical shearing of chromatin using ultra sonic sound waves. The use of this approach is advantageous as it results in random fragmentation of the DNA and is suitable for difficult to lyse cell types or those in which cross-linking has made it difficult for enzymes to access its sites. However, this method requires expensive equipment and can potentially damage epitopes. Typically, sonication for 30 sec on/off for a total of 15 mins works for shearing, though this must be optimized for each cell type.

Enzymatic Digestion
Enzymatic Digestion typically relies on the use of micrococcal nuclease for digestion. It is a milder treatment and it does not require the use of expensive equipment. However, the fragmentation will exhibit sequence bias. In this case, it is critical to optimize the concentration of enzyme used and adjust the digestion times for optimal shearing.

 

 

Antibody Selection
Choosing an antibody that will work well in ChIP applications and is specific for the target protein is critical for a successful ChIP experiment. As the antibody must recognize the native form of the protein, antibodies used in other applications may not work in ChIP experiments. We recommend using a ChIP-validated antibody in addition to control antibodies such as an RNA polymerase II antibody.

The amount of antibody used is critical for any ChIP experiment. If there is too much antibody, it can lead to non-specific binding whereas too little antibody will not be able to bind all of the chromatin-bound protein resulting in misrepresentation of the antibody enrichment. The end-user can consult the product datasheet for a recommend usage of their ChIP-validated antibody, but the optimal concentration of each antibody should be determined empirically for each experiment.

Immunoprecipitation
Immunoprecipitation of chromatin-bound protein can be performed with Protein A, G, or A/G magnetic or agarose beads. In general, Protein A will exhibit greater specificity for rabbit polyclonal antibodies whereas protein G exhibits a broader specificity. Alternatively, our Go-ChIP-Grade™ kits feature columns containing a solid-phase scaffold developed by Chromatrap for immunoprecipitation, which provides increased cell surface area for antibody binding and reduces nonspecific signals.

 

 

Downstream Analysis and Controls
After the cross-links have been reversed, the purified DNA can be analyzed using qPCR or used to generate libraries for Next Generation sequencing. For qPCR analysis, we recommend designing primers to amplify a 100-250 bp region around the gene or binding site of interest to test alongside a positive control primer targeting a known binding site.

In addition to using positive and negative control primers, an input sample should be saved prior to performing the immunoprecipitation so that the enriched DNA can be normalized to the total amount of chromatin used in the experiment. Typically, 1-5% of the starting chromatin is used.

To calculate the percent signal relative to input, use the following calculation:

  1. Adjust Ct of Input sample to 100%.
         For a 5% Input control, subtract 4.32 (log2 of 20) cycles from the experimentally determined Ct value.
  2. Calculate the percent input of each sample including isotype controls.
         Use the equation % input = 100*2^(Adjust Input Ct- Sample Ct)

 

Chromatin Preparation

Step 1: Crosslinking
(Stabilization of Protein-DNA complexes) with 1% formaldehyde, and glycine solution for quenching

Step 2: Cell Lysis
with Hypotonic Buffer

Step 3: DNA Fragmentation by Enzymatic Digestion
with Digestion buffer, lysis buffer, PIC, shearing cocktail, enzymatic stop solution, DNA sample quality and quantity checking

Immunopreciptation

Step 4: Chromatin Immunoprecipitation
with Go-ChIP-Grade™ Purified Antibody, Isotype Control Antibody

Step 5: Column Conditioning, Washing and Elution
with wash buffers and elution buffer

Step 6: Reverse Crosslinking
with 1M NaHCO3, 5M NaCl

Downstream DNA Analysis

Step 7: Digestion with Proteinase K and DNA Purification
with Proteinase K, Proteinase K stop solution, DNA purification cols and buffers

Step 8: qPCR to quantify DNA
with primers to the target gene of interest

Step 9: Data Acquisition and Analysis
(The amount of IP'ed DNA in each sample is represented as signal relative to the 5% of total amount of input chromatin)

Tips for a successful ChIP assay:

  • Always keep your chromatin on ice unless otherwise indicated as it can degrade very quickly and avoid freeze/thaw cycles
  • Make fresh fixation solution and optimize fixation times to ensure that your cells have not been over-fixed
  • Optimize lysis conditions. The number of cells used will dictate the amount of lysis buffer necessary. For hard to lyse cells, you may want to use a homogenizer to efficiently lyse the cells
  • Choose an appropriate shearing method and ensure that the chromatin has been sheared to a size of 150-900 bp. Shearing conditions will need to be optimized for your cell type and amount of chromatin input.
  • Use a ChIP validated antibody with a positive and negative control antibody to ensure that your methodology and experimental conditions were sufficient
  • Use an excess antibody to chromatin ratio, the amount of antibody should be determined empirically

FAQs


How do I know if my chromatin has been sheared?

Purify the DNA from your sheared chromatin samples and run the DNA on a 1-2% agarose gel. DNA should be sheared to a range of 100-900 bp. Below is an example of chromatin from a number of different cells lines that has been sheared optimally using enzymatic digestion compared to chromatin which was over-digested.

 

 

 

Lane 1: Ladder, Lane 2: HeLa, Lane 3: Jurkat, Lane 4: NCCIT, Lane 5: HeLa IL-6

 

Lane 1: HT1080+ IFNγ, Lane 2: NIH353, Lane 3: HT29, Lane 4: HCT116, Lane 5: Ladder

 

Which points in the protocol can I stop and save my experiment?

Sheared chromatin may be saved at -80°C prior to performing the immunoprecipitation. If you are saving the chromatin, we would recommend aliquoting the chromatin as it can be sensitive to freeze thaw cycles. Alternatively, purified DNA can be saved prior to downstream analysis.

Should I use sonciation or enzymatic shearing?

Sonication may be ideal for difficult to lyse cell types and provides random fragmentation but may damage epitopes. It is not suitable for Native ChIP experiments (in which the chromatin has not been cross-linked). Enzymatic digestion is milder and can be used in Native ChIP experiments but will exhibit sequence bias and may not be suitable for hard to lyse cells.

What cell types have been validated with the Go-ChIP-Grade™ kit?

Human and mouse cell lines such as HeLa, Jurkat, Daudi, THP-1, NIH3T3, and 293T have been validated using this protocol. Other cell types (plant, yeast, tissue, FFPE samples, etc.) may require optimization.

How do I determine optimal number of cells for ChIP assay?

An important consideration when performing ChIP is the amount of chromatin that will need to be loaded to the column in order to elute sufficient IP’ed DNA for downstream analysis. Sufficient cell numbers should be used so that at least 3µg of chromatin can be used per IP. Start with 1-15 million cells. However, the cell number can be scaled up and the reagents need to be adjusted accordingly.

How much antibody for target protein should be used per ChIP?

This should be determined empirically by the end-user. Refer to the datasheet for our Go-ChIP-Grade™ Purified Antibodies, or other ChIP-validated antibodies. The suggested dilution of the Go-ChIP-Grade™ anti-RNA Polymerase II Antibody; Clone 8WG16 (positive control) is 1:300 – 1:500 by volume.

What is causing high background?

The quality of the ChIP antibody has a major impact on the success of the assay. Use only ChIP-validated antibodies. Make sure to have good quality chromatin samples with fragments between 150-900bp. Insufficient DNA shearing leads to longer DNA fragment length that can cause high background in downstream experiments. Insufficient wash steps can also leave traces of non-specific chromatin alongside enriched DNA. If background remains high, include an additional wash step during the IP protocol.

Why do I not have any enrichment?

Make sure to only use ChIP-validated antibodies and follow the instructions for the amount of antibody to be used or titrate the antibody for your experiment. It is also recommended to include ChIP validated positive and negative controls (included in the kit) to validate the efficiency of your ChIP assay. Good quality chromatin samples and chromatin fragment sizes between 150 – 900 bp are also critical. Other factors can include whether cells were effectively lysed, over-fixation and reduced antibody binding due to decreased epitope availability, and insufficient starting material (chromatin sample per IP).

 

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