Protein Protocols Protein Protocols

46 Friedrich et al.
2. Materials
2.1. Cells and Antibodies
The CV-1 line of African Green monkey kidney cells was obtained from the American
Type Culture Collection (ATTC, CCL-70). Confluent cultures of CV-1 were
infected with SV40 strain RH-911 at 100 plaque-forming units per cell.
Pab101 is a monoclonal antibody specific to the carboxy (C)-terminus of SV40 T
Ag. Hybridoma cells producing Pab101 were obtained from ATCC (no. TIB-117).
Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were obtained
from Antibodies Inc. (Davis, CA). Alexa conjugated secondary antibodies were
obtained from Molecular Probes (Eugene, OR).
2.2. Solutions for Fixation and Staining
1. Ca2+ /Mg2+ -free phosphate-buffered saline (PBS). Dissolve 8.0 g of NaCl, 0.2 g of KCl,
1.2 g of Na2HPO4 and 0.2 g of KH2PO4 in 1 L of distilled water (dH2O). Adjust the pH to
7.4, sterilize by passing through a 0.2-µm filter and store at room temperature.
2. Trypsin-ethylenediaminetetraacetic acid(EDTA): Dilute 10X trypsin/EDTA (Gibco,
Rockville, MD) in PBS to give final concentrations of 0.05% trypsin, 0.53 mM EDTA.
Filter-sterilize and store at 4°C.
3. Methanol: Store at –20°C.
4. Wash solution (WS): Heat inactivate 100 mL of normal goat serum (Gibco) at 56°C for
1 h. Mix with 900 mL of PBS, 200 µL of Triton X-100, and 1.0 g of sodium azide. Filtersterilize
and store at 4°C.
5. Propidium iodide (PI): Dissolve 1.0 mg of PI (Calbiochem) in 100 mL of PBS. Add 20 µL
of Triton X-100 and 0.1 g of sodium azide. Protect solution from light and store at 4°C.
6. Ribonuclease: Dissolve 100 mg of RNase A (Sigma) in 100 mL of PBS. Boil for 1 h. Add
20 µL of Triton X-100 and 0.1 g of sodium azide. Store at 4°C.
3. Methods
3.1. Fixation
1. Remove culture media and rinse cells with PBS. If floating and/or mitotic cells are of
interest, media and wash fractions should be saved, pelleted, and pooled with attached
cells after trypsinization.
2. Prewarm trypsin/EDTA to 37°C and add 1 mL per 60-mm dish. (Adjust proportionally for
larger culture areas). Tilt plate to thoroughly distribute solution and then remove.
3. Once cells have detached (see Note 1), add 1 mL of WS to plate and transfer cells to a
1.5-mL microcentrifuge tube. Remove a small volume for determination of cell number
(see Note 2). Centrifuge remaining cells at 2000g for 15 s.
4. Discard supernatant, resuspend pellet in 1 mL of cold PBS, and centrifuge as in step 3.
5. Discard supernatant and thoroughly resuspend cell pellet in 0.1 mL of cold PBS.
6. Immediately add 0.9 mL of methanol (–20°C) (see Notes 3, 4), mix, and store at –20°C
(see Note 5).
3.2. Titration of Antibodies
It is important that the primary antibody (see Note 6) be present in excess to ensure
quantitative measurement of the specific protein (see Note 7). Yet, the background of
nonspecific antibody staining should be kept to a minimum. To determine the appropriate
antibody concentration, stain parallel samples of cells with serial dilutions of