실험 Q&A Immunology > Immunoprecipitation
ChIP assay 애물단지네요 ㅠㅠ
레벨2 chip
input조차도 안나와서 무진장 애를 먹고있습니다. sonication조건이 중요하다고들 하셔서 저희방에 있는 sonicator를 사용한 protocol을 참고로 오늘 해봤습니다. size를 먼저 확인하고나서 진행하려고 소니케이션 후 50ul정도 따서 revers-crosslinking ~ PCR purification kit로 DNA purify 한 후에 agarose gel에 걸어봤습니다. 그런데 아무것도 없는거에요 ㅠㅠ 제가 사용한 protocol인데, 한번 봐주시길 부탁드립니다. 1. cells were washed 2. cross-linked with 1.1% formaldehyde at room temperature for 10 min 3. the reaction quenched by addition of glycine to a final concentration of 0.125 M. 4. Cells were harvested, lysed in 10 mM Tris HCl pH 8.0, containing 10 mM EDTA , 0.5 mM EGTA and 0.25% Triton X-100 solution for 30 min at 4°C 5. centrifuged at 200g for 5 min 6. the nuclear pellet washed in 10 mM Tris-HCl pH 8.0 containing 1 mM EDTA, 0.5 mM EGTA and 200 mM NaCl 7. nuclei were resuspended in sonication buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 0.5 mM EGTA, 1% SDS w/v, 1 mM AEBSF, 10 μg/ml aprotinin, and 0.8 μg/ml leupeptin) 8. After centrifugation, the OD260 was adjusted to 6 AU/ml in IP buffer (NaCl 140mM, Triton X-100 1% wt/vol, DOC 0.1% wt/vol, PMSF 1 mM, yeast tRNA 100 mg/ml, BSA 100 mg/ml) 9. precleared for 15 min at 4°C with Staph A cells. 10. incubation with antibodies가 붙은 bead로 O/N 11. bead wash with RIPA 5번 12. antibody/protein/DNA complexes were eluted with 50 mM NaHCO3, 1% (w/v) SDS, and treated with RNase A. 13. Samples were incubated at 67°C for 4-5 hrs 14. The DNA was purified using the QIAGEN PCR Purification kit (Qiagen Inc.,Valencia, CA, USA), stored in 50 μl of 10 mM Tris-HCl pH 8.5, and 2 μl was used for PCR.
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