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Posted by Erbay Yigit on Dec-08-2008.
Depends on what type of molecule you want to isolate (protein or DNA), you need to follow a suitable protocol.
After lysing cells with your favorite method, remove DNA by DNAse I digestion. Mix one volume of protein sample with one volume of 2x Tris-Glycine SDS sample loading buffer. Then, add beta-mercaptoethanol at a final concentration of 2.5%. Heat samples at 96°C for 30 min on a PCR machine or heat block. If you do not remove DNA, you may have some DNA still cross-linked to protein, and you may get a smear in your Western Blots. I have noticed that 30 min heating is sufficient to reverse cross-linking for some DNA binding proteins, but not for others. You may want to increase temperature or duration of incubation for complete uncrosslinking.
If your goal is to run an SDS-PAGE gel, you can simply take harvested cells, mix with 2x Tris-Glycine SDS Sample Loading Buffer, heat it up, and load the gel. In this case you do not need to bother lysing the cells by other methods.
Mix equal volume of sample with 2x ChIP Elution buffer. Add Pronase from 20 mg/ml stock solution at a concentration of 0.8 mg/ml. Incubate 2 hr at 42°C, followed by 6 hr at 65°C. Alternatively, you can use Proteinase K to digest proteins, followed by 6 hr incubation at 65°C. You can also incubate samples at 65°C over night if you want. (2x ChIP Elution Buffer: 50 mM Tris.HCl pH 7.5, 10 mM EDTA, 1% SDS).
Virginia A. Spencer and James R. Davie. The Protein Protocols Handbook, 2nd Edition: Isolation of Proteins Cross-linked to DNA by Formaldehyde. (Totowa, NJ: Humana Press Inc., 2002). Pages 753-757.