University of Virginia Health System

Preparation of whole cell homogenates of cultured mammalian cells

This protocol has been used to prepare protein samples suitable for isoelectric focusing as described in Protocol 17. Different parts of the protocol are designed to accomplish, in order, thorough lysis and solubilization of the cell, fragmentation of DNA, and separation of the proteins from salt and DNA fragments.

Reagents.

1. Tris buffer. 3.0 g tris base (FW 121) + 0.3 g anhydrous MgCl2 are dissolved in approximately 40 mL water. The pH is adjusted to 7.8 with HCl and the final volume adjusted to 50 mL.

2. Lysis buffer. 30 mg SDS + 300 mg DTT are dissolved in 0.6 mL of the tris buffer and the volume adjusted to 10 mL.

3. RNAse: 10 mg/mL RNAse A (Boehringer Mannheim, cat # 109142 from bovine pancreas) prepared in water. Stored at -20°C for up to 1 yr.

4. DNAse: 10 U/µL DNAse I (Boehringer Mannheim, cat # 1776785) purchased as a 10 U/µL solution and stored at -20°C for up to 1 yr.

5. DNAse/RNAse reagent. 20 µL DNAse + 20 µL RNAse + 160 µL tris buffer.

6. Solubilization buffer: 2.1 g urea + 0.8 g thiourea + 200 mg CHAPS + 40 mg DTT taken to 5 mL with water. Heat at 37°C as needed to dissolve urea

We typically collect several pellets from a given system and use one pellet for a Lowry protein assay to assign the amount of protein in each pellet. The procedure below assumes at least 2 mg of protein in a sample but it is preferable to have at least 10 mg. Several pellets may be combined to produce a suitable amount of protein in one sample.

1. Preparation of cell pellet:

a. Culture media is poured off the cells and the cell monolayer is washed with a volume of an ice-cold isotonic buffer such a PBS.

b. A second volume of ice-cold isotonic buffer is added to the dish, the cells harvested by scraping and transferred to a polypropylene centrifuge tube.

c. Cells are pelleted by centrifugation and the supernate poured off. The cell pellets are stored at -20°C for up to 1 yr prior to analysis.

2. Cell pellets are thawed at room temperature and disrupted by vigorous vortex.

3. 1000 µL of lysis buffer are added to the pellets. If several pellets are being combined, then divide the 1000 µL among the pellets and combine in one tube. Mix the sample by trituration with a 1000 µL pipet. At this point, the sample may appear quite viscous and insoluble DNA and RNA may be visible.

4. Heat the samples to 80°C to 90°C in a water bath for 5 min.

5. Cool to RT.

6. Add 10 µL of the DNAse/RNAse reagent. With trituration, insoluble DNA and RNA should appear to solubilize and viscosity should be reduced. If viscosity is still a problem, add a second aliquot of the DNAse/RNAse reagent.

7. Incubate the samples for 5 to 10 min.

8. Add 5 mL of acetone to precipitate the proteins. Vortex mix and cool on ice 10 min.

9. Pellet proteins by centrifugation at 500g for 5 min.

10. Pour off the acetone and air dry the protein pellet 20 to 30 min.

11. Dissolve the protein pellet in solubilization buffer and store at -70°C until analysis. We refer to these samples as our stock samples and routinely prepare these stocks at 10 mg protein/mL. These samples are amenable to isoelectric focussing as described in Protocol 17.