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How do you reverse crosslinking?

Published in Crosslink Reversal 2 mins read

To reverse crosslinking, you can use glycine to effectively terminate the cross-linking reaction. This method is commonly employed in protocols such as Chromatin Immunoprecipitation (ChIP) where the crosslinking of proteins to DNA needs to be reversed to recover the DNA.

The Principle of Crosslinking Reversal

Crosslinking, often induced by agents like formaldehyde, creates covalent bonds between molecules, such as proteins and nucleic acids. These bonds need to be reversed to separate the molecules for downstream analysis. Glycine works by quenching unreacted aldehyde groups from formaldehyde and can also reverse the Schiff base adducts formed during crosslinking.

Practical Steps for Reversing Formaldehyde Crosslinking

In a laboratory setting, specifically within a chromatin immunoprecipitation (ChIP) protocol, the reversal of formaldehyde-induced crosslinking typically follows these steps:

  1. Initial Crosslinking: Cells or tissues are first treated with formaldehyde to induce crosslinking, stabilizing protein-DNA interactions. For example, 37% formaldehyde is added to the culture medium.
  2. Incubation: The crosslinking reaction is allowed to proceed for a specific duration, usually at room temperature.
  3. Crosslink Termination: Glycine solution is then added to stop the crosslinking process and initiate the reversal.

Here's a detailed example of the termination and reversal process, adapted from a standard laboratory protocol:

Step Action Concentration/Amount Temperature Duration Purpose
1 Add formaldehyde to culture medium (if starting from crosslinking) 550μl 37% formaldehyde into 20ml culture medium RT 10 min Induce crosslinking
2 Add glycine solution 125mM glycine solution RT 5 min Terminate and initiate reversal of crosslinks
  • Note: The initial formaldehyde addition step is part of the crosslinking process, but the crucial step for reversal is the addition of glycine.

For more detailed laboratory protocols on crosslinking and reversal, including specific volumes and timings for various experimental setups, you may refer to comprehensive guides such as those found on specialized ChIP protocol resources.

This process ensures that the crosslinks are effectively reversed, allowing for the accurate recovery and analysis of the target molecules.