Recently, I have some troubles in formadehyde crosslinking followed by immunoprecipitation assay. Actually, my experimental purpose is to see whether a protein factor(s) could form a complex with other protein component(s) through a specific cis-element of a target mRNAs of my interest. To do so, I've modified the assay mentioned above, and then got some positive results at the first trial. However, it seemed to go wrong. In some trials, I could see specific RNA-mediated interaction, but it could not in other trials.
During troubleshooting, I've doubt about whether using cold PBS instead of warm PBS during the crosslinking step could affect overall efficiency.
Is it very critical to use warm PBS during the crosslinking step? please give me your valuable advices.
As far as I am concerned, cold PBS is used during the crosslinking step. Crosslinking using formaldehyde is the most critical step during IP, so you should check Formaldehyde concentration you are using for cross-linking. I usually follow Abcam protocol and it works fine with me. I have attached the same here. It recommends to use 0.75% formaldehyde to serve the purpose (i.e. 202 ul of 37-41% formaldehyde). Good luck
Hi Incheol, crosslinking of primary amine residues by formaldehyde is a chemical reaction that is accelerated with increased temperature.
That being said, if you are using high concentrations of formaldehyde (> 0.5%) and incubating for ~30 min, then the temperature effect will be negligible.
Just make sure that the Formaldehyde your using is methanol-free. Methanol is normally added to formaldehyde as a preservative. The effect of added methanol is that it massively increases cells permeability and allow for a much faster crosslinking. Some people have reported that using methanol-containing formaldehyde resulted in in over-crosslinking within 2 minutes
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