Zymo-Spin™ ChIP Kit

Unique workflow features a micro-elution (≥6 µl) spin column for purification of ChIP DNA.
High quality ChIP DNA is ideal for ChIP-qPCR, ChIP-Seq, and other molecular applications.

Product Size Catalog # Price Qty
Zymo-Spin™ ChIP Kit 10 preps D5209
$201.00
Zymo-Spin™ ChIP Kit 25 Preps D5210
$412.00

About Zymo-Spin™ ChIP Kit

The field of epigenetics has grown tremendously over the past several decades. Studies have shown that chromatin modifications, DNA methylation, and DNA hydroxymethylation play critical roles in the regulation of gene expression, gene silencing, protein-DNA interactions, and other cellular processes. Dysregulation of epigenetic modifications can contribute to developmental abnormalities, neurological disorders, and even cancer.

Chromatin immunoprecipitation (ChIP) is the prevailing method used for the study of protein-DNA interactions and the dynamics of epigenetic modifications. ChIP facilitates the identification of regions of the genome associated with a specific protein.  The Zymo-Spin ChIP Kit from Zymo Research provides a streamlined ChIP procedure for investigating protein-DNA interactions that have been “fixed” in their natural state and can be used to effectively identify binding sites for transcription factors, co-factors, and other DNA regulatory proteins.

Briefly, this ChIP protocol involves covalent cross-linking of protein-DNA complexes with formaldehyde followed by cell lysis and chromatin shearing. A ChIP-grade antibody (user supplied) is used with Protein A magnetic beads to immunoprecipitate the protein-DNA complexes of interest.  Following reverse crosslinking, RNase A and Proteinase K treatments, the DNA is eluted in a minimal volume of buffer using a unique micro-elution spin column, eliminating the need for messy precipitations.  The protocol has been optimized for efficient crosslinking, shearing, and immunoprecipitation regardless of the mammalian cell type.  Additionally, eluted ChIP DNA is ideal for end point PCR, quantitative PCR, ChIP-Seq library preparation, and Next-Gen sequencing-based applications.


Equipment Sonicator, magnetic tube rack, vortex, tube rotator, microcentrifuge, agarose gel apparatus/Bioanalyzer/TapeStation, qPCR instrument.
Compatibility This kit contains reagents optimized for mammalian cells.
User Supplied Reagents Formaldehyde (37% stock, w/v), Glycine (2.5 M), Phosphate Buffered Saline, Protease inhibitor cocktail, Phenylmethylsulfonyl Fluoride (0.1 M), Ethanol (95% or 100%), ChIP-grade antibody, QuestTaq™ qPCR Premix

Outline of the Zymo-Spin™ ChIP Kit Procedure

ChIP DNA Purification Method Zymo Research Phenol-chloroform Supplier Q
Total Time 5 min. >60 min. 5 min.
ChIP DNA Purification Comparison
ChIP assays were performed with HeLa cells and ChIP-grade anti-H3K4me3 and rabbit IgG. Both total and immunoprecipitated chromatin were reverse cross-linked and recovered using either the ChIP DNA Clean & Concentrator™ (included in the Zymo-Spin™ ChIP Kit), phenol-chloroform extraction, or DNA recovery kit from Supplier Q. The amount of ChIP DNA was determined using qPCR with primers specific to the GAPDH promoter. ChIP DNA enrichment is graphed as % input (i.e., the relative amount of immunoprecipitated DNA compared to input DNA following qPCR analysis).

SECTION 1 and SECTION 2 below describe formaldehyde cross-linking and chromatin shearing, respectively, with the input of 5 x106 cells. This cellular input will be sufficient for 5 ChIP reactions (Note: If working with more cells, scale up accordingly).

SECTION 1: Cell Collection and Formaldehyde Cross-linking

  1. Harvest cells using appropriate method (e.g., trypsin) and then wash cells twice with 1X PBS.
  2. Count cells and resuspend 5 x106 cells in 1 ml of 1X PBS.
  3. Cross-link cells by adding formaldehyde (user provided) to a final concentration of 1% (v/v) (e.g., add 27 µl of a 37% formaldehyde solution to 1 ml cell suspension).  Incubate for 7 min at room temperature with gentle shaking or rotating.
  4. Stop the cross-linking reaction by adding glycine (user provided) to a final concentration of 0.125 M (e.g., add 50 µl of 2.5 M Glycine to 1 ml cell suspension) and continue shaking or rotating for 5 min.
  5. Important! For Steps 5 to 20, all buffers, tubes, and centrifugation steps must be kept at ~4 °C. 

  6. Centrifuge cross-linking reaction at 3,000 x g for 1 min at 4 °C.
  7. Discard supernatant and resuspend cell pellet in 1 ml chilled 1X PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF)1 and 1X protease inhibitor cocktail (PIC)2 (e.g., add 10 µl of 0.1 M PMSF and 10 µl of 100X PIC to 1 ml chilled 1X PBS).  
  8. Note: Additional protease inhibitors can be added depending on the application: For histone acetylation targets add 10 mM sodium butyrate (HDAC inhibitor) and other inhibitors can be added if required.

  9. Centrifuge at 3,000 x g for 1 min at 4 °C and repeat Steps 6 & 7.
  10. Discard the supernatant and continue with Section 2:Nuclei Preparation and Chromatin Shearing.
  11. Optional: Cross-linked pellets can be stored at -80 °C for future use. 

    SECTION 2: Nuclei Preparation and Chromatin Shearing 

  12. Resuspend the cross-linked cell pellet in 500 µl chilled Nuclei Prep Buffer containing 1 mM PMSF and 1X PIC (e.g., add 5 µl of 0.1 M PMSF and 5 µl of 100X PIC to 500 µl chilled Nuclei Prep Buffer).
  13. Vortex briefly to ensure cell pellet is completely resuspended and incubate on ice for 5 min.
  14. Centrifuge at 3,000 x g for 1 min at 4 °C.
  15. Discard supernatant and resuspend cell pellet in 500 µl chilled Chromatin Shearing Buffer containing 1mM PMSF and 1X PIC (e.g. add 5 µl of 0.1 M PMSF and 5 µl of 100X PIC to 500 µl chilled Chromatin Shearing Buffer).
  16. Important! Do not vortex, resuspend cell pellet by pipetting.

    Note: If you noticed a precipitate in Chromatin Shearing Buffer, warm the buffer briefly by hand until the precipitate is dissolved.

  17. Incubate on ice for 5 min.
  18. Sonicate on ice for 4 cycles (30 sec “ON”, 30 sec “OFF” at 40% amplitude). Tip must be submerged (~¾ inch) in the buffer to avoid foaming2.
  19. Centrifuge at 12,000 x g for 5 min and then transfer the supernatant containing the sheared chromatin (~500 µl) to a pre-chilled 1.5 ml tube.
  20. SECTION 3: Immunoprecipitation with ZymoMag Protein A

    This protocol has been optimized for 100 µl inputs of sheared chromatin (~1 x106 cells) per 1 ml ChIP reaction.  If working with fewer cells, scale down the volumes proportionally.

  21. Prepare the following ChIP reaction mixes for both the target antibody and negative control antibody1:
  22.               100 µl      Sheared Chromatin

                       x µl      ChIP-grade Antibody2

              880 - x µl      Chromatin Dilution Buffer

                    10 µl      100X PIC3

                    10 µl      0.1 M PMSF

                      1 ml     Final Volume

    Important! Be sure to set aside 10 µl of the sheared chromatin for use as a DNA input control.  Keep DNA input control at 4 °C.

  23. Incubate ChIP reactions 3 h to overnight at 4 °C while rotating.
  24. Add 15 µl ZymoMag Protein A4 beads into the ChIP reactions and incubate for 1 h at 4 °C while rotating.
  25. Place tubes on a magnetic stand, allow the beads to cluster, and then remove and discard the supernatants.
  26. Wash the Protein A bead/antibody/chromatin complexes by resuspending the beads in 1 ml each of the chilled buffers in the order listed below:
  27. Chromatin Wash Buffer I, 1st wash, 1 ml

    Chromatin Wash Buffer II, 2nd wash, 1ml

    Chromatin Wash Buffer III, 3rd wash, 1ml

    Incubate for 4 min while rotating between each wash followed by magnetic separation with careful removal of supernatants.

  28. Resuspend beads in 500 µl DNA Elution Buffer (do not confuse with 5X Chromatin Elution Buffer) and transfer the bead suspension to a clean 1.5 ml microcentrifuge tube to avoid co-elution of protein that may be bound to the tube wall.
  29. Place tubes on magnetic stand, allow the beads to cluster, and remove the supernatant.
  30. SECTION 4: Elution and Reverse Cross-linking of ChIP DNA

  31. Dilute the 5X Chromatin Elution Buffer with water to obtain a 1X Chromatin Elution Buffer.
  32. Note: If you notice a precipitate in the 5X Chromatin Elution Buffer, simply warm at

    37°C until the precipitate is dissolved.

  33. Resuspend beads in 150 µl of 1X Chromatin Elution Buffer, gently mix by pipetting.  
  34. Important! Add 140 µl of 1X Chromatin Elution Buffer to 10 µl of the sheared chromatin

    (DNA input control) from Step 16.

  35. Add 6 µl 5 M NaCl and mix by gently flicking the tube.
  36. Incubate at 75 °C for 5 min and centrifuge at ≥ 10,000 x g for 30 seconds.
  37. Place tubes on magnetic stand, allow the beads to cluster, and then transfer eluates (ChIP DNA) into clean 1.5 ml microcentrifuge tubes.
  38. Incubate at 65 °C for 30 min.
  39. Add 1 µl Proteinase K and incubate at 65 °C for 90 min.
  40. SECTION 5: ChIP DNA Purification

    Buffer Preparation: Before starting, add 24 ml 100% ethanol to the 6 ml DNA Wash Buffer concentrate to obtain working DNA Wash Buffer solution.

  41. Add 5 volumes of ChIP DNA Binding Buffer to each volume of DNA sample (5:1) and mix briefly (e.g. add 750 µl of ChIP DNA Binding Buffer to 150 µl ChIP DNA or input DNA sample).
  42. Transfer mixture to a provided Zymo-Spin IC Column in a Collection Tube.
  43. Centrifuge at ≥ 10,000 x g for 30 seconds.  Discard the flow-through.
  44. Add 200 µl DNA Wash Buffer to the column.  Centrifuge at ≥ 10,000 x g for 30 seconds.
  45. Repeat wash step (Step 30).
  46. Transfer the column to a new 1.5 ml tube and add 8 µl DNA Elution Buffer directly to the column matrix. Let stand for 1 minute at room temperature and centrifuge at ≥ 10,000 x g for 30 seconds to elute the DNA.
  47. Repeat elution (Step 32). Total elution volume for both ChIP DNA and input DNA samples is 16 µl.
  48. Use immediately or store ChIP DNA at -80 °C

 


For specific notes and additional information, please see the product protocol PDF.
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Featured Citations

Researchers utilized the Zymo-Spin™ ChIP Kit to further establish an epigenetic signature of rheumatoid arthritis (RA). They identified the Limb Bud and Heart Development (LBH) gene as a novel transcriptional enhancer that may contribute to the aggressive nature of RA.
Researchers studying craniofacial and dental development at the University of Iowa used the Zymo-Spin ChIP Kit to demonstrate the Tbx1 regulation of miR-96 thru the binding of Tbx1 at 3251 base pairs upstream of the miR-96 transcription start site. This is the first demonstration of Tbx1 repression of a miRNA.
The authors showed the binding of the transcription factors Sp1, c-Myb and NFI to the most upstream α9 negative regulatory element by using the Zymo-Spin ChIP Kit. The data helps to further characterize α9β1, which is a recent member of the integrin family of membrane receptors.
ChIP assays were performed using the Zymo-Spin™ ChIP Kit to assess the enrichment of active transcription markers (i.e. histone H3ac and RNA polymerase II) across the Brain-Derived Neurotrophic Factor (BDNF) gene. The greater enrichment of these active transcription markers at BDNF gene in cells expression HTLV-1-encoded protein, HBZ provided evidence that the HBZ is involved in transcriptional regulation of BDNF.

“An excellent product that is easy to use.”
A. A. – Karmanos Cancer Institute
“The kit reduced the amount of time needed for my ChIP experiments, the DNA purification was more efficient than other kits, and the purity of the DNA was higher. The endpoint results generated were highly informative to my experimental objectives.”
F. E. – Cleveland Clinic
“The microelution was key here. The most significant problem we have faced in the past with performing ChIP on primary cells is the number of cells required to obtain useable data. This assay far exceeded our expectations and will allow us to perform routine analysis using relatively small numbers of primary cells.”
K. B. – RML/NIAID/NIH
"It is good there is DNA Clean-up & Concentration Kit."
X. L. – University of Cincinnati
"Kit materials are excellent."
B. P. – Thomas Jefferson University
"Thank you for the opportunity in using this affordable and easy to use kit; it was slightly different from the previous I used, but I agreed in minutes to the new protocol, with enhanced yields and results."
V. I. – 'Sapienza' University of Rome
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