I have been trying to perform CoIP experiments on two proteins with transient interactions by using formaldehyde as a cross-linking reagents. However, I am experiencing a significant amount of HEK 203T cell loss after my incubation with formaldehyde and glycine.
Currently I co-transfect my two proteins of interest into 10 cm dishes. During the cell harvesting:
1. Aspirate growth media off.
2. Wash two times with 3 mL of warm 1X PBS
3. Incubate with 5 mL of 4% formaldehyde for 10 minutes with minor rocking at RT.
4. Wash two times with 3 mL of ice-cold 1.25 M glycine.
5. Add 1X whole cell lysis buffer with protease inhibitor and rock at 4 degrees for 30 minutes
I believe I am losing cells during the excessive washing steps from 2 to 4. Is it possible to recover the cells via centrifugation? Or should I adopt another protocol that is more efficient at preserving the cells?
Hi Rong Xuan Zang . I would have an issue with step 4. You dont need 1.25M glycine to neutralize the formaldehyde, 3 washes with TBS should be enough.
In step 3, you could increase the fixation time to 30 min with no rocking at RT.
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