ChIP sequencing (ChIP-Seq) is a powerful method for identifying genome-wide DNA binding sites for transcription factors and other proteins. Use this guide to help troubleshoot common issues that arise during ChiP procedures:
- Pre-clear lysate sample. Clear lysate with protein A/G affinity beads to remove proteins that nonspecifically bind and increae background signal.
- Use fresh buffers Contaminated buffers can cause increased background. Prepare fresh lysis and wash buffers to eliminate this source of error.
- Use high-quality protein A/G beads. Low-quality protein A/G beads can give high background signal. Use Boster’s quality-guaranteed protien A/G beads to ensure good results.
- Reduce sonication. Excessive sonication can result in low fragment sizes that produce poor results. Optimize sonication time to yeild fragments between 200-1000 bp.
- Improve cell lysis. Insufficent lysis will result in low signal. Use Boster’s high-quality lysis buffers to ensure good results
- Reduce cross-linking intensity. Excessive cross-linking by formaldehyde fixation can mask epitopes and reduce signal intensity. Reduce fixation time and quench with glycine to increase signal.
- Use more starting material. Too little starting material will yeild poor results. We recommend using 25 ug of chromatin per immunoprecipitation.
- Use more antibody. Increase amount of antibody used to boost signal. We recommend between 1-10ug of antibody to maximize results.
- Reduce salt concentration of wash buffers. Wash buffers with excessive osmolarity can reduce antibody binding activity. We recommend using buffers with no more than 500 mM salt.
Low Resolution and High Background
- Reduce DNA fragment size. Optimize sonication to acheive fragment length of 200-1000 bp.