Currently I am doing RNA immunoprecipitation to find the targets of an RNA binding protein. I am unclear about the reverse-crosslinking protocol. I have found two approaches one in which they wash with 2M urea after immunoprecipitation and incubating at 70 °C for 45 min, whereas in the other protocol they just incubate at 70 °C for 45 min? Kindly help me with the detailed protocol on de-crosslinking step involved in RIP assay. Thank you.
The reverse crosslinking or de-crosslinking step is essential for separating the RNA from the RNA-binding protein (RBP) after immunoprecipitation. Here, I provide you with a detailed protocol for the de-crosslinking step in RIP assay:
After the final wash step of your RIP assay, carefully remove as much wash buffer as possible without disturbing the beads.
Add 100-200 µL of reverse crosslinking buffer to the beads. The composition of the reverse crosslinking buffer is:
50 mM Tris-HCl (pH 7.0)
5 mM EDTA
10 mM DTT
1% SDS
Note: You may need to adjust the volume of reverse crosslinking buffer depending on the size of your pellet.
Resuspend the beads by pipetting up and down or by gentle vortexing. Ensure that the beads are properly resuspended in the buffer.
Incubate the sample at 70 °C for 45 minutes with occasional mixing (e.g., vortexing briefly every 10-15 minutes). This step will help reverse the formaldehyde-induced crosslinks between the RNA and the RBP.
Following incubation, centrifuge the samples at maximum speed (≥14,000 x g) at 4 °C for 5 minutes to pellet the beads.
Carefully transfer the supernatant, which contains the released RNA, to a new tube without disturbing the beads. Proceed to RNA isolation and purification using your preferred method (e.g., phenol-chloroform extraction, RNA purification kit).
The two protocols you mentioned differ in the use of urea. The 2M urea wash can help denature proteins and disrupt protein-protein interactions, which can be helpful in some cases to improve the recovery of RNA. However, it's not strictly necessary for the de-crosslinking step. The protocol I provided above does not include the urea wash, but if you would like to incorporate it, you can perform a wash with 2M urea in RIP wash buffer after the final wash step and before adding the reverse crosslinking buffer.
Thank you for your timely input and suggestions Dr. Arup. I will try to incorporate this de-crosslinking step in my experiment. Thank you.
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