4 Steps to Better ChIP results - Step 1- Take control

Posted by Carolyn P on Feb 18, 2015 3:00:00 AM


You're a confocal maestro, playing the lasers and the filters and the gain settings like a Stradivarius to generate those eye-catching, data-filled images that turn your peers green with envy.  You sit back, work a cross-word puzzle, and watch with confidence while the red and green lights dance down your screen. But what happens when you reach the slide with that random treatment your advisor insisted you include (over your sound objections) . . . and you find your favorite cytoplasmic protein homing into the nucleus like the graduate students after free pizza?

You'll make a bee-line for your advisor, thinking "I may have to eat a little crow, but it's totally worth it." And, right as you're about to knock on his/her door it will hit you - oh, no! - I'm going to have to figure out how to do ChIP. 

Don't worry - we can help!

Chromatin Immunoprecipitation (ChIP) is used to examine interactions between protein and DNA within the natural chromatin context of the nucleus. ChIP experiments first require fixing the cells, which cross-links the protein-DNA interactions into place. The chromatin is then broken into fragments and an antibody is used to immunoprecipitate the protein of interest along with any bound DNA. Finally, the cross-linking is reversed and the precipitated DNA is purified. The purified DNA can be subjected to further analysis, such as standard or real-time PCR, microarray, or sequencing.

These experiments are sensitive to the integrity of the chromatin, the quality of the protein epitope and the specificity of the immunoprecipitating antibody; and these variables become even more critical when the protein-DNA interaction under investigation occurs rarely or is unstable.


Topics: Protocols, ChIP, techniques

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