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| 1. |
Suspend cells mixed with PBS buffer in ice-cold lysis buffer1. |
| 2. |
Sonicate for several minutes. |
| 3. |
Spin the lysate for 15 minutes at 14,000 x g with a pre-cooled centrifuge. |
| 4. |
Transfer the supernatant to a fresh tube. |
| 5. |
After washing with PBS buffer twice, keep protein A/G agarose beads in 50% slurry state in PBS (use blunt-tipped pipette in this procedure). |
| 6. |
Add 10 ㎕ of protein A/G slurry into 1 ㎖ of cell lysate and incubate for 1hour at 4℃ to pre-clear cell lysate. |
| 7. |
Spin the lysate for 1 minute at 3,000rpm at 4℃ to remove protein A/G slurry. |
| 8. |
Transfer the supernatant to a fresh tube. |
| 9. |
Mix adequate amount of immunoprecipitation antibody with 200 ㎕ pre-cleared cell lysate(1 ㎎/㎖). |
| 10. |
Pour lysis buffer to total amount of 500 ㎕. |
| 11. |
React cell lysate/antibody mixture for 2 hours at 4℃ on an orbital shaker. |
| 12. |
After adding protein A/G slurry 10ug to capture immune complex, react for 2 hours at 4℃. |
| 13. |
Spin for 1 minute at 3,000rpm at 4℃. |
| 14. |
Remove the supernatant. |
| 15. |
Wash out protein A/G slurry with lysis buffer three times, spin for 1 minute at 3,000rpm at 4℃. |
| 16. |
Mix beads well with 20ul of 1x sample buffer. |
| 17. |
Boil protein A/G beads for 5 minutes to separate immune complex. |
| 18 |
Separate beads by centrifuge and perform SDS-PAGE, immunoblotting with the supernatant (see SDS-PAGE, immunoblotting).m |
