Guides to cell number for ChIP and endpoint analysis

ChIP can be performed on either cultured cells or tissues. In either case, it is important to consider the homogeneity of your sample, as each contributing cell type may have a different chromatin structure. It is also important to determine how many ChIP reactions you will be performing. The number of cells required for each ChIP reaction will depend on the abundance of the protein target. Factors to consider when deciding the “cell equivalents of chromatin“ required per ChIP reaction include the quantity of available epitopes in your cells as well as the quality (affinity and specificity) of your antibody. It is possible to achieve successful enrichment with as few as 1 X 10e4 cells per ChIP, if you are using high quality antibodies against abundant epitopes, such as RNA polymerase II and some types of histone modifications. We recommend increasing the amount of cells when the antibody is less optimal or the number of epitopes per cell is low.

Tables 1 and 2 provide guidelines for the number of cells required for each type of protein target. You should scale cell numbers according to the performance of your antibody to optimize highest signal-to-noise ratio relative to a nonspecific control (normal IgG or negative-location control). Tissue samples contain heterogeneous cell populations and can be especially challenging in ChIP. You may use either cryosectioned or fresh tissue microdissected into 1-3 mm3 sections. The amount of tissue you use for ChIP will also depend on the type of tissue, the relative abundance of your protein target, and the robustness of your ChIP antibody. As a guideline, a pea-size piece of tissue contains approximately 10e7 cells and should be sufficient for 100 ChIP samples. The accuracy of this value, however, depends on the relative cellularity of your tissue and the amount of contaminating extracellular matrix. You should always handle tissue samples carefully and promptly, keeping samples on ice to preserve the sample integrity.

Table 1: Guide to cell numbers for ChIP

Abundance of
protein target
Molecules
per locus
Cells/ChIP
Examples
High High 104 Modified histones, RNA Pol II
Medium Medium 105-106 General  transcription  factors, modifiers (TFIID, PCG)
Low Low 106-107 Sequence-specific transcription  factors
Low Indirect binding 107 or more Accessory factors

 

Table 2: Guide to cell numbers for endpoint analysis

Application
Required
Number of Cells
Application
qPCR Picogram-nanogram 105 qPCR
ChIP-seq ~1-10 nanogram 106 ChIP-seq
ChIP-chip ~10-100 nanogram 108 ChIP-chip

For more information about ChIP experiment guides, troubleshooting tips and supplementary protocols, please view our Chromatin Immunoprecipitation (ChIP) page.