Analysis of Proteins

2.2.6.1 Protein Isolation from Mammalian Cells

Cells were grown in 10 cm dishes, and harvested when they reached confluency of 70%.

The Medium was aspirated and cells were washed 3 x with ice-cold PBS. Then the cells were incubated with TNE lysis buffer for 30 min on ice. The lysed cells were scraped off the plates, transferred into microcentrifuge tubes and centrifuged for 15 min at 14,000 x g at 4°C. The Supernatants were removed, and pellets were discarded. An equal volume of 2 x SDS Sample buffer was added to each sample, the samples were boiled for 10 min, then chilled on ice and microcentrifuged for 5 min. Aliquots were stored at –20°C until use.

TNE lysis buffer

10 mM Tris-HCl, pH 7.8

150 mM NaCl

1% NP-40

1 mM EDTA

1/20 ml protease inhibitor complete ^TM tabs

2 x SDS Sample buffer

50 mM Tris-HCl, pH 6.8

100 mM DTT

2% w/v SDS

10% w/v Glycerol

0.1% w/v bromophenol blue

Prior to Western Blot analysis of caspases, an alternative protocol was used for the preparation of cellular extracts. The cells were allowed to grow for 48 hours then the medium was aspirated, transferred into pre-chilled Falcon tubes, and kept on ice. The cells were washed 3 x with ice-cold PBS and scraped into PBS. Then the tubes with medium and cells in PBS were centrifuged for 5 min at 1,000 x g. The supernatants were discarded, and CHAPS cell extract buffer (one volume of cell pellet) was added to the cell pellets.

The cells were resuspended in CHAPS buffer, frozen and thawed three times to lyse them, and centrifuged at 14,000 x g for 15 min at 4°C. The Supernatants were transferred into fresh tubes, an equal amount of SDS-Sample buffer was added, boiled for 10 min, and chilled on ice. The aliquots were stored at -20°C until use.

Chaps cell extract buffer

50 mM Pipes/NaOH, pH 6.5

2 mM EDTA

0.1% Chaps

5 mM DTT

20 µg/ml Leupeptin

10 µg/ml Pepstatin

10 µg/ml Aprotinin

1 mM PMSF

2.2.6.2 Subcellular Fractionation

Cells were seeded in 10 cm dishes and allowed to grow in their culture medium until they were 70% confluent. The medium was aspirated, cells were washed 3 x with ice-cold PBS, and incubated with hypotonic-lysis buffer (1 ml per 10 cm plate/2 x 10⁶ cells) for 5-10 min on ice. Samples were transferred into Dounce Homogenizers, pre-chilled on ice and homogenised by repeated strokes to disrupt the cells. To pellet the nuclei cells were centrifuged at 1000 x g for 10 min at 4°C. Supernatants were transferred into Beckman tubes (13 x 15 mm), the pellets containing nuclei were saved on ice.

To recover membrane-associated proteins, the supernatants were centrifuged in a TLA 100.3 rotor at 100 000 x g for 30 min at 4°C. The supernatants containing cytoplasmic proteins were removed and transferred into Corex tubes, pellets were dissolved in 50µl ice-cold PBS and saved on ice.

To collect cytoplasmic proteins, a methanol/chloroform precipitation was used (Wessel and Flugge, 1984). To each supernatant prepared in the previous step, 4 ml pre-cooled methanol was added, then 1 ml pre-cooled chloroform was added and mixed, then 3 ml pre-cooled sterile water was added and mixed. The solution was centrifuged at 9 000 x g for 15 min at 4°C. The upper phase was discarded, and 3 ml methanol were added to each sample, mixed and centrifuged again 9 000 x g for 15 min at 4°C. The pellets were dried and dissolved in SDS sample buffer.

SDS-sample buffer was added to all fractions, samples were boiled for 10 min, chilled on ice, and stored at -20°C until use.

Hypotonic lysis buffer

10 mM Tris-HCl, pH 8.0

0.1 mM DTT

1 tab/20 ml protease inhibitor complete ^TM tabs 2.2.6.3 Determination of Protein Concentration

The protein concentration was determined using the BSA Protein Assay (PIERCE) according to the recommendations of the supplier.

2.2.6.4 One-Dimensional SDS Gel Electrophoresis (PAGE)

Protein samples were separated by polyacrylamide gel electrophoresis (PAGE). 50 µg of protein extract for detection of H-REV107-1 protein, and 30 µg for detection of caspases were loaded on a 12 % gel, run 30 min at 65V, and 2,5 hours at 98V in 1 x running buffer.

Separating gel: 10 ml of a 12% gel

Stacking gel: 10 ml of 4% gel

6.1 ml H2O

After gel electrophoresis was finished, the stacking gel was discarded, and the separating gel was immersed in transfer buffer for 15 min. A PVDF-membrane was immersed in methanol for 5 sec, then in water for 2 min with shaking on a rotor platform, then the membrane was incubated 15 min in transfer buffer. On a Bio-Rad Transblot 3 sheets of Whatman paper pre-wetted in transfer buffer, the PVDF membrane, the gel, 3 sheets of pre-pre-wetted Whatman were assembled.

Proteins were transferred to the PVDF membrane during 35 min at 16 V. After the transfer was finished, the membrane was washed briefly in TBST buffer, and blocked for 1 hour in 15 ml of blocking solution at room temperature with shaking on a rotor platform.

The gel was stained in Comassie blue solution to control for equal loading of protein amounts and efficient transfer. Afterwards the gel was photographed and discarded.

After the blocking procedure, the membrane was washed 3 x 10 min in 15 ml TBST, and incubated overnight with the primary antibody in blocking solution. The next day the membrane was washed 3 x 10 min in 15 ml TBST, and incubated for 1 hour with the corresponding secondary antibody.

The membrane was washed again 3 x 10 min in TBST, and the signal were developed using an ECL kit (Amersham), according to the supplier’s recommendations.

To strip a membrane, a Western Blot Recycling kit (Alpha Diagnostic) was used. Membranes were incubated in stripping solution for 45 min up to 1 hour, and rinsed with blocking solution supplied with the kit 2 x 5 min. After an additional washing in TBST for 10 min the membrane was ready for re-probing with other antibodies.

Primary antibodies, corresponding secondary antibodies, and their dilutions

Primary antibody Dilution Secondary antibody Dilution Anti-cleaved caspase-3 (Cell

1:10 000 peroxidase-conjugate goat anti-mouse (Dianova, Hamburg,

Anti-p21 (Santa Cruz

1: 5000 peroxidase-conjugate goat anti-mouse (Dianova, Hamburg,

1: 500 peroxidase-conjugate goat anti-rabbit (Dianova, Hamburg,

Anti-V5 (Invitrogen, CA, USA) 1 : 5000 peroxidase-conjugate goat anti-mouse (Dianova, Hamburg,

Blocking buffer 5% non-fat dry milk in TBST

Commasie blue 0.1% Comassie blue R-250 dissolved in 40% Methanol, 10% acetic acid.