Hi, I am currently trying to co-immunoprecipitate a protein but the interaction between that protein and its binding partner is not stable. Sometimes we able to detect its binding partner but sometimes we don't. So, I plan to do formaldehyde cross-linking for my HEK293T cells. Can anyone share with me their protocol? For example, how many formaldehyde should I use and how long should I treat the cells?
Any opinion is welcome and thanks to you in advance.
This may work if you incubate the cytosol, not the whole cells as they would become difficult to disrupt. You would have to remove the formaldehyde before addition of antibody (perhaps with a desalting column). Concentration of formaldehyde and incubation time you'd have to optimise for signal strength and background, I'd start with those used for fixing cells (4% and a few minutes).
Yes, I am using pFA to cross-link the protein complex. It works with 0.1% pFA for 8min at RT, then add glycine to the final concentration of 125 mM to quench the reaction. You can also titrate the pFA concentration best for your IP. After this you can lyse the cells and go for IP. To reverse the cross-linking you may have to boil your material in the SDS-PAGE sample buffer for 20min. Please let me know if you need more info
As Arunasalam recommended above, you could follow fixation steps used for ChIP from any standardized protocol. Crosslinking (Avoid excessive crosslinking) is a critical step for certain proteins and the purity of PFA used. For example, in Page 18, https://docs.abcam.com/pdf/chromatin/A-beginners-guide-to-ChIP.pdf
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