CST BLOG: Lab Expectations

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Increase Your Experimental Success with CUT&RUN-Validated Antibodies

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CUT&RUN delivers the power of chromatin immunoprecipitation (ChIP) or ChIP-sequencing (ChIP-seq), only takes one to two days to go from cells to DNA, and can be used with as few as 5K to 20K cells. Like any antibody-based technique, identifying a compatible antibody is a critical part of ensuring assay success. CUT&RUN is a relatively new technique, limiting the number of validated antibodies for the application. Therefore, some companies recommend using ChIP- or ChIP-seq-validated antibodies for CUT&RUN since all three techniques are used to analyze protein-DNA interactions across the genome in cells and tissues.

But do all ChIP-validated antibodies really work in a CUT&RUN assay? In fact, we have found this not to be the case. Let's explore why.

Why ChIP- or ChIP-seq-Validated Antibodies Might Not Work With CUT&RUN

Even though both ChIP and CUT&RUN assays are used to profile chromatin, there are significant differences between the methodologies that can impact antibody recognition. A summary of these differences is provided in Table 1.

 

ChIP

CUT&RUN

Sample Preparation

Cross-link chromatin with formaldehyde and lyse cells.

Bind cells to Concanavalin A-coated magnetic beads and permeabilize cell membrane with digitonin.

Chromatin Enrichment

In vitro immunoprecipitation using an antibody specific for a DNA binding protein or histone modification.

In vivo enrichment, where the antibody specific for a DNA binding protein, cofactor, or histone modification enters the nuclei after cell membrane permeabilization.

Chromatin Fragmentation

Non-targeted enzymatic digestion with MNase or mechanically using sonication to shear the chromatin prior to chromatin immunoprecipitation.

Targeted enzymatic digestion during enrichment based on the interaction of a primary antibody with pAG-MNase.

Chromatin/Protein State

Fragmented chromatin containing partially denatured histones and DNA-binding proteins.

Contiguous chromatin with histones and DNA binding proteins in their native conformations.

Table 1: ChIP and CUT&RUN methodologies. 

Not all ChIP- and ChIP-seq-Validated Antibodies Work in CUT&RUN

We tested antibodies for over 100 (and counting!) target proteins and found that only 50-60% of ChIP- or ChIP-seq antibodies were compatible with CUT&RUN. ChIP experiments were run using the SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, while CUT&RUN experiments were performed using the CUT&RUN Assay Kit #86652 and DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209. Downstream quantitative polymerase chain reaction (qPCR) analyses were performed using SimpleChIP Universal qPCR Master Mix #88989 and DNA library preparation was done using the DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 and Multiplex Oligos for Illumina (Dual Index Primers) #47538. NG-seq was performed using an Illumina NextSeq Platform.

Related: A Guide to Successful ChIP & ChIP-seq

When Stat1 (D1K9Y) Rabbit mAb #14994, which is ChIP-validated, and Stat1 (D4Y6Z) Rabbit mAb #14995, which is ChIP- and ChIP-seq-validated, were tested, both antibodies demonstrated robust enrichment of target genes in ChIP (Figure 1A). Stat1 #14994 did not generate passable data for ChIP-seq, but it did show enrichment in the CUT&RUN assay. On the other hand, Stat1 #14995, which is validated for ChIP and ChIP-seq, did not work for CUT&RUN since there was no observed enrichment for known STAT1 target genes IRF1 or AIM2 (Figure 1B).

22-ETC-39681 C&R 2.0 Blog_Social Fig1Figure 1. ChIP and CUT&RUN experiments were performed on HT-1080 cells treated with Human Interferon-γ (hIFN-γ). While both STAT1 rabbit monoclonal antibodies show robust enrichment of target genes in ChIP (A), only Stat1 (D1K9Y) Rabbit mAb #14994 shows target gene enrichment in CUT&RUN (B). Note that while the Stat1 (D4Y6Z) Rabbit mAb #14995 is validated for and shows target gene enrichment in ChIP-seq, it does not work for CUT&RUN (B).

Three Stat3 antibodies were also tested: Stat3 (124H6) Mouse mAb #9139 and Stat3 (79D7) Rabbit mAb #4904, which are both ChIP- and ChIP-seq-validated, and Stat3 (D3Z2G) Rabbit mAb #12640. Again, all three antibodies robustly enriched target genes using ChIP (Figure 2A), but only Stat3 #9139 demonstrated robust target gene enrichment with CUT&RUN (Figure 2B). Stat3 #12640 did show some target gene enrichment in CUT&RUN, but ultimately did not meet Cell Signaling Technology (CST) validation criteria because a low genomic signal-to-noise ratio was observed.

22-ETC-39681 C&R 2.0 Blog_Social Fig2-1Figure 2. ChIP and CUT&RUN experiments were performed with HepG2 cells starved overnight and treated with IL-60. While all three STAT3 monoclonal antibodies show robust enrichment of target genes in ChIP (A), only Stat3 (124H6) Mouse mAb #9139 shows robust target gene enrichment in CUT&RUN (B). Note that while the ChIP-seq-validated Stat3 (D3Z2G) Rabbit mAb #12640 does show enrichment of target genes in CUT&RUN (B), it failed validation due to high background signal.

Antibody

ChIP Validated

ChIP-seq Validated

Works on CUT&RUN

Stat1 (D1K9Y) Rabbit mAb #14994

Yes

No

Yes

Stat1 (D4Y6Z) Rabbit mAb #14995

Yes

Yes

No

Stat3 (124H6) Mouse mAb #9139

Yes

No

Yes

Stat3 (79D7) Rabbit mAb #4904

Yes

No

No

Stat3 (D3Z2G) Rabbit mAb #12640

Yes

Yes

No

Table 2: Summary of described antibodies tested on CUT&RUN.

As these two examples illustrate, relying solely on the fact that an antibody is validated for ChIP or ChIP-seq does not necessarily guarantee CUT&RUN success. Therefore, the best bet is to use a CST CUT&RUN-validated antibody to ensure success. However, you can start with ChIP- or ChIP-seq-validated antibodies if a CUT&RUN-validated antibody hasn’t been identified yet. Just be aware that you’ll likely need to screen different antibodies to find one that will work.

To save you time and money that you’d otherwise spend testing antibodies yourself, CST is actively validating CUT&RUN antibodies for you!

How CST Validates Antibodies for CUT&RUN

CST is known for having stringent validation standards to ensure antibodies will work for your application, and it’s no different when it comes to validating antibodies for CUT&RUN. To provide antibodies that are proven to work in CUT&RUN, we test all of our antibodies using the CUT&RUN Assay Kit #86652, the DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209, the SimpleChIP Universal qPCR Master Mix #8898, the DNA Library Prep Kit for Illumina #56795 (ChIP-seq, CUT&RUN), and the Multiplex Oligos for Illumna (Dual Index Primers) #47538.

Antibodies must pass all of the following criteria to qualify as a CUTRUN-validated antibody:

  1. Antibody sensitivity: Compare the target enrichment signal-to-noise ratio to input controls. The antibody must provide an acceptable minimum number of defined enrichment peaks and a minimum signal-to-noise ratio compared to input chromatin to pass.
  2. Antibody specificity:
    • Compare the signal from knock-out cell lines with wild-type cells. If knock-out cells are not available, look for a consistent profile of enrichment peaks across the genome using multiple antibodies against distinct target protein epitopes.
    • Look for a consistent profile of enrichment peaks using antibodies raised against different subunits in a multiprotein complex.
    • Compare enrichments across the genome to published CUT&RUN and ChIP-seq data (i.e., ENCODE) using additional antibodies for a given target protein.
    • Analyze enrichment of known positive and negative target loci using qualitative PCR (qPCR). The antibody must show a minimum fold enrichment for the known positive loci compared to the known negative target loci. It also must show a minimum signal-to-noise ratio determined by the level of enrichment of the known target loci by the target-specific antibody vs. an isotype control antibody.
  3. Antibody specificity for sequence-specific DNA-binding transcription factors: Perform transcription factor binding-motif analysis of enriched chromatin fragments.

Using ChIP- or ChIP-seq-validated antibodies for CUT&RUN does not automatically guarantee success since only 50-60% of them are compatible. Before you spend time screening antibodies yourself, be sure to check our ever-growing list of CUT&RUN antibodies validated by CST. You may be able to save yourself valuable time!

Additional Resources:

CST and Cell Signaling Technology are trademarks or registered trademarks of Cell Signaling Technology, Inc. All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information. 22-ETC-39681
Christopher Fry, PhD
Christopher Fry, PhD
Chris Fry, PhD, is a Senior Director of Epigenetics, Assays, and Workflow Solutions at Cell Signaling Technology. He is an epigenetics scientist trained at the University of Wisconsin and UMass Medical Center. At CST, he is helping to develop and manage the epigenetics product portfolio with a specific interest in developing antibodies and antibody-based tools to investigate epigenetic mechanisms in disease.

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