Preparation of Cell Lysate used for Immunoprecipitation
Extracts prepared from the isolated nuclei of cultured cells can be used in functional studies and for the purification of proteins. This protocol adapted from Mirmira Lab, University of Virginia outlines the steps and processes required.
Recipes
Phosphate Buffered Saline (PBS), pH 7.2
| NaCl | 8.0 g |
| KCl | 0.2 g |
| NaH2PO4 | 0.23 g |
| Na2HPO4 | 0.12 g |
- Adjust the volume to 1 liter with distilled water.
- Store at 4-25 °C.
IP Lysis Buffer
| NaCl | 7.31 g |
| 1 M Tris, pH 8 | 25 ml |
| 0.5 M EDTA, pH 8 | 5 ml |
| 10% NP-40 | 25 ml |
| Distilled Water | 445 ml |
- Protease Inhibitors (Calbiochem Cat#539131)
- Reconstitute with 1 ml of dH2O. Use 50 mcl per 5 ml of lysis buffer to harvest cells.
- Store at 4 °C.
Procedure
- Allow cells to grow to approximately 80% confluence on 100mm polystyrene tissue culture plates. Approximately 8*106 cells (one plate at 80% confluence) are needed per IP reaction.
- Wash cells two times in cold PBS.
- Lyse cells to release soluble cellular proteins using 500 mcl of cold lysis buffer containing 1X protease inhibitors (Calbiochem, Cat. No. 539131) per 100 mm plate. Scrape cells from the plates, transfer to 1.5 ml micro-centrifuge tubes, and place on ice for 30 minutes to ensure efficient lysis.
- Centrifuge lysates at 10,000 x g; 5 minutes.
- Remove and pool the supernatant. Determine protein concentration.
