Protein chemical methods

2.2.17.1 Protein isolation from HeLa cells

Nuclear proteins from HeLa cells were specifically isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer’s instruction. First, the medium were removed and cells were washed twice with DPBS and then harvested using TrypLE Express (Invitrogen). To neutralise the trypsin, DMEM medium was added instead of DPBS and the cell suspension was centrifuged at 500 x g for 5 minutes. The supernatant was removed

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and the cell pellet washed by suspending in DPBS. The cell suspension was centrifuged again at 500 x g for 5 minutes. The supernatant was discarded and the cell pellet suspended in 1.5 ml DPBS and then transferred into a 1.5 ml e-cup.

Following centrifugation steps were performed at 4 °C, cell samples and extracts were kept on ice. Next, the cells were centrifuged at 500 x g for 2-3 minutes. The supernatant was carefully removed to leave the cell pellet as dry as possible.

Depending on the cell volume the reagent volume was determined as shown below.

Table 36: Reagent volumes for different packed cell volume

Packed Cell Volume (µl) CER I (µl) CER II (µl) NER (µl)

10 100 5.5 50

20 200 11 100

50 500 27.5 250

100 1,000 55 500

Right before using CER I and NER protease inhibitors (100 x stock) were added to maintain extract integrity and function. First, ice-cold CER I was added to the cell pellet which was then fully suspended by vortexing on the highest setting for 15 seconds. After 10 minutes incubation, the appropriate amount on ice-cold CER II was added. The tube was vortexed for 5 seconds on the highest setting, then incubated for 10 minutes and vortexed again for 5 seconds on the highest setting.

Samples were centrifuged for 5 minutes at maximum speed (~ 16,000 x g). The supernatant consisting of the cytoplasmic extract was transferred to a new prechilled tube. To the pellet which contains the nuclei, the appropriate amount on ice-cold NER was added. For a total of 40 minutes the tube was vortexed every 10 minutes for 15 seconds on the highest setting and then centrifuged for 10 minutes at maximum speed (~ 16,000 x g). The supernatant which contains the nuclear extract was transferred to a new prechilled tube. The protein concentration was determined by Synergy Mx spectrophotometer (BioTek). Extracts were aliquoted and stored at

80 °C.

2.2.17.2 Measurement of protein concentration

After the principle according to Bradford (1976), the concentration of proteins was measured. Therefore, Roti^®-Nanoquant (Roth) was employed as described by Witt

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(2013). This solution contains Coomassie Brilliant Blue that binds under acidic conditions to proteins which leads to a conversion of the red form to the blue form of the dye causing a shift in the absorption spectrum maximum from 365 nm to 595 nm (Bradford, 1976). A standard protein, namely BSA (0-6.7 µg BSA/ml), was used for calibration. Based on the standard curve the protein concentration could be extrapolated. A triple measurement of each sample was performed and the average of the values was determined. First, a 1:100 dilution of the desired protein extract was prepared. 50 µl of the diluted protein extract and 50 µl of ddH2O as a reference were put in a 96 well plate and 200 µl of the Roti^®-Nanoquant solution was added. After 5 minute incubation at room temperature, the absorption was measured at 590 and 450 nm by Synergy Mx spectrophotometer.

2.2.17.3 Co-IP

The Co-IP is a technique used to analyse protein-protein interactions. With an antibody a known protein is targeted which is believed to be a member of a protein complex, the protein antigen is precipitated as well as proteins that are bound to it.

By western blot analysis and further protein detection methods it might be possible to identify unknown complex members. For Co-IP nuclear cell extract was used and the immunoprecipitation of the target protein was carried out using the Immunoprecipitation Kit (Protein G) from Roche according to the manufacturer’s instructions with modifications regarding incubation times, number of washing steps and during centrifugation steps. Depending on the amount of nuclear cell extract, an appropriate amount of the specific antibody was added and the sample incubated on a rotating platform overnight at 4 °C (Roche protocol: 1 hour). The next day 45 µl of the protein G was added to a new tube and centrifuged for 1 minute at 13,000 rpm at room temperature (Roche protocol: 50 µl). The supernatant was removed and the overnight protein solution was added to the remaining protein G. Next, the mixture was incubated on a rotating platform for 2 hours at 4 °C (Roche protocol: 3 hours).

Complexes were collected by 3 minutes centrifugation at 3,000 rpm at 4 °C (Roche protocol: 12,000 x g for 20 sec). After the supernatant was removed, the beads were washed using 500 µl of the lysis buffer (Roche protocol: 2 times 1 ml lysis buffer and 20 minutes incubation). The samples were centrifuged again for 3 minutes at 3,000 rpm at 4 °C (Roche protocol: 12,000 x g for 20 sec). The beads were resuspended in 1 ml wash buffer 2 and centrifuged under the same conditions as

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before (Roche protocol: centrifugation at 12,000 x g for 20 sec after 20 minutes incubation). After the supernatant was removed, a final washing step using 1 ml wash buffer 3 was performed (Roche protocol: 2 times 1 ml wash buffer 3 and 20 minutes incubation). The following preparation to load the sample on an SDS gel was performed differently. The supernatant was removed after centrifugation and 18 µl DPBS (PAN), 18 µl LDS (Invitrogen) and 4 µl 1M DTT were added to the beads. To denaturise proteins and separate them from the protein G beads, the mixture was incubated for 5 minutes at 95 °C. Beads were centrifuged down and the supernatant was loaded on an SDS gel and further analysed by western blot (2.2.17.5).

2.2.17.4 SDS polyacrylamide gel electrophoresis

By SDS polyacrylamide gel electrophoresis (SDS PAGE) proteins are denaturised and can be separated according to their molecular weight in a polyacrylamide gel by an electric field (Shapiro et al., 1967; Laemmli, 1970). In this work 4-12 % Bis-Tris Gels (Invitrogen) and 3-8 % Tris-Acetate Gels 3-8 % Tris-Acetate Gels (Invitrogen) were used for protein separation (2.2.17.5).

2.2.17.5 Western blot

The western blot is an analytical technique and this method was used to transfer proteins from an SDS gel to a nitrocellulose membrane where the proteins can be detected by different reactions (Towbin et al., 1979; Towbin and Gordon, 1984;

Towbin et al., 1992).

Protein extracts used for Co-IP were incubated with 18 µl DPBS (PAN), 18 µl LDS (Invitrogen) and 4 µl 1M DTT for 5 minutes at 95 °C. After spinning down the Protein-G-agarose beads, 30 µl of the supernatant which contains the precipitated protein along with other proteins bound to it in a denaturised form was loaded on an SDS gel. Two different transfer methods were used in this work.

a) semidry

With this transfer variant proteins < 100 kDa were transferred from an SDS gel to a membrane. Using MES SDS Running Buffer (Invitrogen) and a 4-12 % Bis-Tris Gel (Invitrogen), proteins were separated according to their molecular weight. For approximately 2.5-3 hours the gel was running at 100 V. The protein transfer was

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performed according to Wieczerzak (2012). The blotting condition was changed to 220 mA instead of 230 mA. 4 Whatman Papers which were soaked in transfer buffer were placed on the Turboblotter^TM (Schleicher & Schuell). A nitrocellulose membrane which was wetted in ddH2O and transfer buffer was put on top of it followed by the SDS gel and another 4 prewetted Whatman Papers. Approximately 50 ml transfer buffer were needed. The blotting was performed at constant 150 mA for 15 minutes followed by 45 minutes at 220 mA.

b) wet blot

This transfer method is appropriate for larger proteins (> 100 kDa). First proteins were separated according to their molecular weight using gel electrophoresis.

Therefore, Acetate SDS Running Buffer (Invitrogen) and 3-8 % Tris-Acetate Gels (Invitrogen) were used. The running time of the gel amounted 3-3.5 hours at 100 V.

To prevent heating of the buffer, the gel chamber was placed in an ice box. Once the proteins were separated, the “gel-sandwich” was assembled in transfer buffer to avoid bubbles according to BIO-RAD (n.d.). On the grey coloured side of the cassette (Bio-Rad) a foam pad was placed followed by 2 Whatman Papers, a nitrocellulose membrane and the SDS gel. Finally, 2 Whatman Papers and another foam pad were placed on top. The cassette was closed and placed vertically between parallel electrodes in the buffer tank (Bio-Rad) containing a frozen blue cooling unit and the transfer buffer (Tab. 13). To avoid the formation of an ion gradient and to insure the temperature homogeneity, a magnetic stir bar was placed in the buffer tank which was put in a box filled with ice on a magnetic stirrer. For the blotting at 4 °C, the voltage was set on 60 V constant for 2.5 hours.

2.2.17.6 Protein detection using antibodies

Once the protein transfer from the SDS gel to the membrane was completed, the membrane was transferred into a falcon tube (CellSTAR) and unspecific binding sites were blocked by incubating the membrane in TBST with 5 % milk or 5 % BSA for 1-2 hours at room temperature. During incubation steps membranes were placed on a rolling platform. The membrane was then incubated overnight at 4 °C with the appropriate primary antibody. Antibodies were diluted 1:1,000 to 1:2,000 in TBST with 2 % milk or 2 % BSA. Next day the membrane was washed three times for 10 minutes in 20 ml TBST with 2 % milk or TBST if BSA was used. The membrane

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was then incubated for 2 hours at room temperature or at 4 °C with a secondary antibody conjugated to horseradish peroxidase (HRP). The antibodies were 1:7,000 to 1:10,000 diluted in TBST with 2 % milk (incubation at room temperature) or 2 % BSA (incubation at 4 °C). After that, the membrane was washed three times for 10 minutes in TBST with 2 % milk and once with TBST for 5 minutes or three times for 10 minutes with TBST at room temperature. To visualise proteins, SuperSignal^TM West Pico Chemiluminescent Substrate (Thermo Scientific) was used according to the company’s instructions. To prepare the substrate working solution, the two substrate components were mixed in a 1:1 ratio (light sensitive). The membrane was placed between a clear plastic wrap and incubated 5 minutes in the dark with SuperSignal West Pico Substrate Working Solution. HRP produces a detectable light signal in the presence of a substrate which can be either detect on X-ray films or by the detecting system FlourChem^® Q (Alpha Innotech).