Protein Protocols Protein Protocols

Preparation of Eukaryotic Cells for 2-DE 161
3. Methods
3.1. 35 S-Labeling (see Note 3)
1. Jurkat T lymphocytes are labeled for 3–24 h in methionine-free media containing 50–250 µCi/
mL of 35 S.
2. Follow cell lysate protocol (Subheading 3.3.).
3.2. 32 P-Labeling (see Note 3)
1. Add 100 µCi/mL of 32 P for up to 3 h to cells that are in phosphate-free medium (see Note 4).
3.3. Whole-Cell Lysate Preparation
1. Wash cells with PBS three times in a microcentrifuge tube.
2. Add an equal volume of hot (100°C) dSDS solution to the pellet.
3. Boil tube (100°C) for 1–3 min.
4. Cool in an ice bath (see Note 5).
5. Add 1/10 volume of DNase/RNase solution.
6. Gently vortex mix for several minutes to avoid foaming. The sample should lose its
viscosity, and the solution should look clear. If not, then add more dSDS and DNase/
RNase solution (see Note 6).
7. Snap-freeze in liquid nitrogen, and store at –70°C. Samples may be kept for up to 6 mo
at –70°C.
3.4. Preparaing the Sample for 2D-PAGE
3.4.1. Vacuum Drying
1. Lyophilize sample (frozen at –70°C) in a Speed Vac using no or low heat until dry.
2. Add SB solution to the sample equal to that of the original dSDS sample volume, and mix
thoroughly.
3. Heat sample to 37°C for a short period if necessary (see Note 7).
4. Store at –70°C. samples can be kept for up to 6 mo at –70°C.
5. Radioisotope incorporation in the sample may now be determined by trichloroacetic
acid (cTCA)precipitation.
6. Recommended first-dimension load is 500,000 dpm for 35S-labeled proteins and
200,000 dpm for 32P-labeled proteins.
7. If necessary, the sample is diluted and mixed thoroughly with SBS solution before loading
onto the first-dimension gel (see Note 7).
4. Notes
1. cGLPs and cGMPs will help with all aspects in a research facility. Industry will have
Quality Assurance/Control departments to oversee the implementation of these practices.
Smaller research labs may wish to assign or hire a person to oversee that these practices
are adhered to.
2. Maintain Jurkat T lymphocytes in complete culture medium supplemented with streptomycin/penicillin,
in a humidified incubator with 95% air and 5% CO2 at 37°C and at a
concentration of 105 –106 cells/mL.
3. It is extremely important that all radioactive work be performed with the utmost care, and
according to your institutional and local guidelines.
4. It may be necessary to preincubate the cells. Preincubating the cells at a density of 1–10 ×
106 cells/mL for 30 min in sodium-free, phosphate-free medium works well.