How to Handle Samples After Adding Crosslinkers in Protein Crosslinking
In experiments analyzing protein–protein interactions via crosslinking methods, cells are commonly treated with chemical crosslinkers such as formaldehyde. Following crosslinker treatment, it is essential to quench the crosslinking reaction. This can typically be achieved by adding Tris buffer, glycine, or by rapidly cooling the cells to 4°C. After crosslinking, the cells may be either flash-frozen for storage or processed immediately for lysis.
For example, when glycine is used to terminate the crosslinking reaction:
1. Following crosslinking, glycine is added to the sample. Glycine reacts with formaldehyde, thereby quenching its crosslinking activity.
2. The cells are then washed multiple times to remove residual glycine and formaldehyde.
3. Cells are collected and resuspended in an appropriate lysis buffer to proceed with downstream analysis. The choice of buffer, such as RIPA or NP-40 lysis buffer, should be guided by the specific requirements of the experiment.
4. Cell lysis is performed using mechanical disruption or ultrasonic homogenization.
5. Finally, the lysate is subjected to centrifugation to remove cellular debris, and the resulting supernatant containing proteins is collected for further analysis.
The steps outlined above represent a commonly used protocol; however, specific procedures may vary depending on the experimental design and objectives.
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