The success of your chromatin immunoprecipitation (ChIP) experiment depends on the fragmentation of chromatin, a critical step in the ChIP protocol. This can be accomplished with either sonication or enzymatic digestion. But how do you decide which chromatin fragmentation protocol to use in your ChIP experiments? A number of factors can influence your choice, making a decision seem daunting. So let’s simplify – watch the video and we'll will show you the way!
Chromatin immunoprecipitation sequencing (ChIP-seq) is a flexible and powerful technique used by researchers to elucidate how gene regulation is involved with different biological events and with the progression of various conditions like cancer and neurodegenerative diseases.
Researchers use chromatin immunoprecipitation, or ChIP, to identify and characterize protein-DNA interactions in the context of chromatin. ChIP experiments can use varying input samples, chromatin fragmentation methods, and provide ChIP-qPCR or ChIP-seq readouts.
It's time to check out another video from the CST Tech Tips playlist! In this edition of Tech Tips, we'll tackle a common protocol question customers ask our ChIP team: how much antibody to use for chromatin immunoprecipitaion (ChIP) experiments. Adding more antibody isn't always better - watch the video to learn why.
Can't see the embedded video above? Click the link below to play:
Don't forget to subscribe to CST's YouTube channel for more videos to help your research!
Researchers who run a lot of chromatin immunoprecipitation "ChIP" assays – maybe even your advisor – might subscribe to the idea that polyclonal antibodies perform better than monoclonal antibodies. But is that always actually true?
It’s worth your time to understand the differences between the two in terms of antigen recognition and specificity, and dispel some myths.
So your experiments and data are funneling you down an inescapable path. You need to show direct gene regulation by your protein of interest. You think to yourself, “Oh, ChIP...”
The success or failure of a chromatin immunprecipitation (ChIP) experiment is highly dependent on the integrity of both the chromatin and the protein epitope, so your method of chromatin preparation can have a significant impact on the quality of your results.
Part one of this series described the importance of including proper controls to your protocols. In Part 2 we discussed how chromatin preparation affects the final outcome of the experiment. Now, let's take a minute to consider the immunoprecipitating antibody.
Antibodies that are not highly specific to the target of interest may bind unpredictably and increase the background noise; and this may make it more difficult to detect less abundant or lower stability interactions.
Part one of this series described the importance of including proper controls to your protocols. Now, we will examine how chromatin preparation affects the final outcome of the experiment.
First consider the type of interaction you are trying to detect:
- High-frequency, very stable protein-DNA interactions like those between histones and DNA, occur frequently enough that they may still be detected even if the protocol is not fully optimized.
- Low-frequency, less stable interactions like the binding of polycomb group proteins to specific genes (e.g., Ezh2), may fall under the detection threshold if the protocol fails to safeguard the integrity of the protein and the DNA, or if it relies on an antibody that is not highly specific to the target of interest.
Now it's time to consider your options for preparing your chromatin...