This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.
- Place cell culture dish on ice and wash the cells with ice cold Phosphate-buffered saline (PBS).
- Drain PBS and add ice cold lysis buffer.
- Scrape cells off the dish using a cell scraper and gently transfer the cell suspension into a microcentrifuge tube.
- Maintain agitation for 30 minutes at 4°C.
- Centrifuge in a micro centrifuge at 4°C. The time and force of centrifugation may vary depending on the cell type.
- Gently remove the tubes from the centrifuge and place them on ice.
- Aspirate the supernatant ad place in a fresh tube on ice.
- Add 100uL denaturing lysis buffer to the cells.
- Mix well by vortexing vigorously for 2 to 3 seconds.
- Transfer the cell suspension to a microcentrifuge tube.
- Heat samples to 95°C for 5 minutes to denature.
- Dilute the suspension with 0.9mL of non-denaturing lysis buffer. Mix gently.
- Fragment the DNA by passing the lysed suspension 5-10 times through a needle attached to a syringe.
- Incubate on ice for 5 minutes.
- Dissect the tissue quickly with clean tools. If possible, do so on ice to prevent degradation.
- Place tissue in microcentrifuge tubes and snap freeze by immersing in liquid nitrogen.
- Add 300uL of lysis buffer for approximately 5mg of tissue and homogenize with an electric homogenizer.
- Rinse twice with another 300uL of lysis buffer per rinse and maintain constant agitation for 2 hours at 4°C.
Note: If denaturing is required, follow steps 2-5 from the denaturing protocol above.
- Centrifuge for 20 minutes at 12000rpm at 4°C in a microcentrifuge. Aspirate the supernatant and place in a fresh tube kept on ice.
Pre-clearing the Lysates
- Add 50uL of normal serum to 1 mL of lysate and incubate for 1 hour on ice.
- Add 100uL of bead slurry to the lysate and incubate for 30 minutes at 4°C.
- Spin in microcentrifuge at 14,000 x g for 10 minutes at 4°C.
- Discard bead pellet and keep supernatant for immunoprecipitation.
Immunoprecipitation with Antibody in Solution
- While on ice, add 10-50ug cell lysates to a microcentrifuge tube plus the recommended amount of antibody.
- Incubate the sample with the antibody for a few hours at 4°C with gentle agitation.
Note: The length of incubation depends on the amount of protein and the affinity properties of the antibody.
- Prepare the Sepharose beads.
- Mix the slurry well and add 70-100uL of the beads to each of the samples, while on ice.
- Incubate the beads mixture for 4 hours at 4°C with gentle agitation.
- Centrifuge tube after incubation and wash 3 times with lysis buffer.
- Add 25-50 uL 2x loading buffer and boil for 5 minutes at 100°C.
- Centrifuge and transfer the supernatant to a new tube for Western blotting.