Simple Protein Crosslinking

Simple Protein Crosslinking

1. Have tagged, purified protein ready before starting the rest of this procedure.
2. Grow up a culture of target cells (500mL is fine).
3. Spin down the culture.
4. Resuspend the culture in nanopure water and lyse by sonication.
5. Spin down the cell debris.
6. Freeze the supernatant and lyophilize.
7. To the dry protein extract add up to 500uL of tagged protein (make sure you have at least 2mg of tagged protein).
8. Add crosslinking buffer at RT, 2mL.
9. Let sit at RT for 15 min and swirl gently every min.
10. Quench reaction with 1.25M glycine.
11. The solution is now ready to be purified with an affinity column.

Crosslinking Buffer:

• 1% paraformaldehyde
• 1x PBS buffer
• pH > 7, aim for 8
• Heat @70C for 1 hour or until paraformaldehyde is dissolved