Hi, I am performing a ChIP protocol. There i start with my cells in dishes, which I fix in FA%, the I quench the FA, lyse them and then I pellet the chromatin. Then I perform the immunoprecipitation using beads and the corresponding antibody and in my protocol the last step requires to separate the proteins from the bead-antibody complex by heating the beads in LB for 3 minutes at 95. I was wondering if that step may in any way affect the crosslinks already? I read that in order to revert them most of the protocols suggest to heat them up around 60 degrees for at least 3-4 hours, also overnight. Thus, could I be reverting the crosslinks already with the 95 degrees 3 minutes incubation? if it may be affecting them, how could I check this?
Additionally, should I have my samples in a specific buffer or conditions in order to reverse the croslinks?
Herewith 2 different methods, have for you for reverse cross link and DNA isolation.
1. Add 100 μl of 10 % DNA Purifying Slurry to the beads and vortex.
2. Incubate at 95°C for 15 min to reverse crosslink.
3. Add 1 μl of Proteinase K and 1 μl of 1 mg/ml RNase A. Incubate at 60°C for 15 min.
4. [Optional]: Incubate at 95°C for 10 min to inactivate the enzyme
5. Phenol chloroform extraction or another DNA purification method can also be used.
(OR)
Elute DNA by adding 120 μL of elution buffer to the protein A/G beads and vortex slowly for 15 min at 30°C.
Centrifuge for 1 min at 2,000 x g and transfer the supernatant into a fresh tube.
Add 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL) and incubate while shaking at 65°C overnight.
Add 2 µL proteinase K (20 mg/mL) and incubate while shaking at 60°C for 1 h.
The DNA can be purified using a PCR purification kit or phenol:chloroform extraction.
From the above methods; 95'C used for reverse cross link and 60'C used for Protein digestion.
Good luck
Hey thanks you very much Bhaskar. Mmm I was wondering, what is the relevance of proteinase K really?
Hey welcome you.
Proteinase K was commonly used to digest protein, and to separate contaminants from DNA.
Okey. I incubated my samples at 95ºC 20 minutes and in parallel 65ºC 4 hours and judging from my agarose gel crosslinks in neither were not removed. I will try now with preoteinase K and Rnase A and see if i can degrade the X-linked factors and hope my DNA runs just determined by the length.. i will update
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