2. MATERIALS AND METHODS
2.2 WESTERN BLOTTING ANALYSIS
2.2.1 Sample preparation from trypsinised cells
In experiments where cells were harvested by trypsinisation and counted, aliquots were retained for analysis by Western blotting. Counted cells were transferred into 1.5ml Eppendorf tubes. Cells pellets were obtained by centrifugation at 1000xg for 3minutes at 4°C and the PBS was aspirated. Cell pellets were lysed in 100µl of Radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitor cocktail (prepared according to manufacturer’s instruction; Roche) for 30minutes on ice, by vortexing with 5minutes intervals for 30seconds. The lysate was then centrifuged for 30minutes at maximum speed (17949.49xg) at 4°C. The supernatants were transferred to fresh 1.5ml Eppendorf tubes and the protein concentrations of supernatants determined with a Pierce Bicinchoninic acid assay (BCA) protein assay kit (Thermo Scientific) as instructed by the manufacturer.
2.2.2 Whole cell protein extraction
To avoid significant cleavage of cellular protein, cell samples were prepared by lysing the cells in situ using RIPA lysis buffer with protease inhibitor cocktail (prepared according to manufacturer’s instruction; Roche). The medium was aspirated, and cells were washed twice in ice cold PBS prior to an appropriate amount of lysis buffer being
added. Cells were incubated on ice for 15minutes before scraping and then transferred to a 1.5ml Eppendorf tube. Lysates were centrifuged for 30minutes at maximum speed (17949.49xg) at 4°C. The supernatants were transferred to fresh 1.5ml Eppendorf tubes and the protein concentrations of supernatants were determined with a Pierce BCA protein assay kit (Thermo Scientific) as explained by the manufacturer.
2.2.3 Quantification of extracted protein
Briefly, 25μl of each sample was incubated for 30minutes at 37°C with 200μl of working solution in a 96-well plate (Corning Costar, UK), in triplicate. Subsequently, the absorbance was measured by a FluoStar spectrophotometer at 562nm. Protein concentrations were calculated by using a standard curve of known albumin standards ranging from 0 to 1500μg/ml. Protein samples were stored at -70°C until analysed. 2.2.4 SDS-PAGE technique
2.2.4.1 Acrylamide gel preparation
The components for the solutions used in the Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) technique are listed in (Table 2-6). Proteins from cell lysates were separated by protein size using SDS-PAGE. The mini- Protean 3 electrophoresis system (Bio-Rad, USA) was used to cast 8-12% acrylamide resolving gels (1.5mm thick). In this study, different concentrations of acrylamide gels were prepared depending on the size of the protein investigated, 10% for E-cadherin and β-catenin, and 12% for smaller sized proteins (
Table 2-7). Gels were allowed to set for at least 30minutes before loading and running. Then a stacking gel was made on top of the resolving gel at 5% concentration (Table2-7).
Table 2-6: Western blotting buffers used in this study.
Buffers used in western blotting and their components and concentrations.
Solution components
10% SDS 10xElectrode buffer
10g Sodium Dodecyl Sulphate
(SDS) 30.3g Tris base
100ml H2O 144g Glycine
1.5M Tris-HCl/ SDS pH8.8 1L H2O
45.41g Tris Running buffer
0.4% SDS (10ml of the 10% stock) 0.1% SDS (10ml of 10% stock) 200ml H2O 1xTris Glycine (100ml of 10xelectrode buffer)
pH to 8.8 with 1M HCl Make up to 1L with H2O
Make to 250ml with H2O Transfer Buffer
1M Tris-HCl/SDS pH6.8 100ml Methanol
6.06g Tris 1XTris Glycine (50ml of 10xelectrode buffer) 0.4% SDS (4ml of 10% stock) Make up to 500ml with H2O
90ml H2O 0.1% Ponceau S Red
pH to 6.8 with 1M HCl 0.1mg
Make to 100ml 1L 1% Acetic acid
Table 2-7: A list of the concentration of resolving SDS/PAGE gels used in this study.
Component volumes (mL) per gel mould volume of 10ml.
Solution components Components volumes (ml) per gel
volume of 15ml Gel concentration 8% 10% 12% H2O 6.9 5.9 4.9 30% Acrylamide mix 4 5 6 1.5M Tris (pH8.8) 3.8 3.8 3.8 10% SDS (SLS, UK) 0.15 0.15 0.15 10% ammonium persulfate 0.15 0.15 0.15
TEMED 0.018 0.016 0.012
Table 2-8: List of the solutions used in the preparation of 5% stacking SDS/PAGE.
Components of solution used to prepare 5% stacking gel. Components volumes (ml) per gel mould volume of 4mL.
Solution components
Components volumes (ml) per gel mould
volume of 4ml H2O 2.7 30% Acrylamide mix 0.67 1.5M Tris (pH6.8) 0.5 10% SDS (SLS, UK) 0.04 10% ammonium persulfate 0.04 TEMED 0.008
100µg of protein (unless otherwise stated) was loaded into each lane. 10µl of Amersham ECL Plex Fluorescent Rainbow Marker (GE Healthcare Life Sciences,
USA) was loaded into a spare well and sample was electrophoresed until stain front
remained visible.
2.2.4.2 Sample preparation
Equal amounts of protein were loaded from each lysate that were diluted in 1x NuPAGE LDS sample buffer (Novex, Life Technologies, USA) or Laemmli sample buffer (Bio-Rad, USA) containing 5% Beta-Mercaptoethanol. Samples were then heated in a preheated heat block at 100°C for 5minutes. The samples are cooled briefly in ice prior to loading into the gel.
2.2.4.3 Gel electrophoresis conditions
Gels were run at constant voltage (125v) for 90minutes in room temperature in 1x running buffer (Table 2-6).
2.2.4.4 Protein transfer
The proteins were then transferred onto polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences, USA) using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, USA). PVDF membrane is activated by incubation in 100% methanol for 15minutes. Then proteins were transferred from gel to
PVDF in methanol containing transfer buffer (Table 2-6) at constant voltage (25V) for 90minutes. To confirm successful protein transfer, both the gel and the membrane were stained by a reversible stain with 0.1% Ponceau S (w/v) in 1% acetic acid (v/v), subsequently removed by washing with distilled water, without affecting the proteins in the blots.
2.2.5 Probing for proteins of interest
2.2.5.1 Blocking, washing and antibody preparation
Membranes were blocked in 5% low fat milk prepared in PBS containing 0.1% Tween 20 (PBS/T) overnight at -4°C with continuous agitation. Membranes were washed in PBS/T three times for 3minutes between incubations. Antibody dilutions (Table 2-9) were prepared in blocking buffer. Loading accuracy was subsequently assessed by analysis using a primary antibody raised against α-Tubulin.
Table 2-9: List of primary antibodies used for probing the membranes.
The primary antibodies used in this study and their origin. Also, dilations and incubation period used in this study.
Antibody Clone Isotype Company Concentration Band size Control
E-cadherin cadherin 34/E- monoclonal Mouse IgG2b Becton, Dickinson (BD) Transduction laboratoriesTM 1:100 120kDa HT29, and MCF7 b-catenin 14/Beta-catenin monoclonal IgG1 Mouse BD Transduction laboratoriesTM 1:150 92kDa
SW480, and HT29 Vimentin RV202 monoclonal IgG1 Mouse BD Transduction laboratoriesTM 1:50 57kDa
MDA-MB- 231, and
Hela Gli1 H-300 Rabbit poly clonal IgG Biotechnology Santa Cruz 1:100 173kDa 114- K562, and PC3 Gli2 C-10 monoclonal IgGMouse
1
Santa Cruz
Biotechnology 1:100 168kDa 86- K562, and PC3
Gli3 B-4
Mouse monoclonal IgG2b kappa light
chain
Santa Cruz
Biotechnology 1:100 190kDa K562, and Jurkat
Smoothened E-5
Mouse monoclonal IgG2a
kappa light chain
Santa Cruz
Biotechnology 1:100 85kDa K562, and Hela
Patched 3B3
Mouse monoclonal IgG2a
kappa light chain
Santa Cruz Biotechnology 1:100 140kDa PC3 Sonic Hedgehog (Shh) E-1 Mouse monoclonal IgG1 kappa light chain
Santa Cruz
Biotechnology 1:100 19-45kDa Hela Indian Hedgehog
(Ihh) H-12
Mouse monoclonal IgG1 kappa light chain
Santa Cruz
Biotechnology 1:100 45kDa Hela, and HepG2 a-Tubulin DM1A monoclonal IgG1 Mouse ALDRICH SIGMA- 1:1000 50kDa -
Table 2-10: List of secondary antibodies used for visualisation of blot and their origin.
Table include a summary of dilation and incubation periods of antibodies.
Antibody Host Reactivity Company Concentration
IRDye® 800CW anti-Mouse IgG (H+L) Donkey Mouse Li-Cor® 1:15000
IRDye® 680RD anti-Rabbit IgG (H+L) Donkey Rabbit Li-Cor® 1:15000
2.2.5.2 Visualisation and imaging of proteins bands
Protein bands were detected using the Enhanced Chemiluminescence (ECL) method by adding 2mL of the chemiluminescent substrate to the membrane (Millipore Forte) for 2minutes in continuous rotation, followed by developing the ECL film (Amersham, GE Healthcare, USA) in the dark room after 30second, 1minute and 3minutes exposure. The Li-Cor Odyssey® FC system was used for detection of secondary antibodies labelled by the near-infrared fluorescence. Images were captured at appropriate exposure time using the appropriate detector filter using the Image StudioTM software.
2.2.5.3 Densitometric analysis
In order to quantify differences in protein expression observed by Western blotting analysis, the films were scanned (600dpi) using Photoshop software (Adobe) and densitometric analysis was performed using ImageJ software (NIH, USA). All densitometric analysis was normalised to a common band on each film and for protein screening experiments, averages are representative of data from three separate experiments.