I am attempting to perform a coIP after co-culturing cells and need to perform reversible crosslinking. I was thinking of using DSP and DTT to perform the reversible crosslinking. Has anyone performed this type of crosslinking on the two different cell lines? Trying to figure out the logistics on how to add the crosslinking reagent to both cells lines in their preferred growing conditions as the DSP cannot be added into the media.
It is possible to perform reversible crosslinking using DSP (disuccinimidyl suberate) and DTT (dithiothreitol) on two different cell lines that have been co-cultured. However, there are a few challenges and considerations to keep in mind when using these reagents in this context.
One challenge is that DSP cannot be added directly to the growth media, as it can react with other components in the media and interfere with cell growth. Therefore, it will be necessary to remove the cells from the growth media and add the DSP to the cells in a separate solution. This can be done by gently washing the cells with a solution containing DSP, allowing the DSP to react with the cells for a specified amount of time, and then washing the cells again to remove any unbound DSP.
Another challenge is that DTT can inactivate DSP, so it will be necessary to add DTT to the cells after the DSP has been allowed to react. This can be done by washing the cells with a solution containing DTT, allowing the DTT to react with the cells for a specified amount of time, and then washing the cells again to remove any unbound DTT.
Overall, while it is possible to perform reversible crosslinking using DSP and DTT on co-cultured cells, it will be necessary to carefully consider the logistics of adding these reagents to the cells in their preferred growing conditions. It may also be helpful to consult with other researchers who have experience with this type of crosslinking to obtain guidance and advice.
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