Immunoblotting

2.12.1 Preparation of whole cell extract

For western blotting when cells reached 60-70% confluency they were seeded in 100mm culture plates. After 24 hours of leaving the plates in 37oC incubator wherever necessary cells were transfected with Polyfect or DharmaFECT transfection reagent, or media was changed to DCC media and cells were treated with appropriate hormones for periods mentioned in each experiment. The media then was removed and cells were washed in 1X PBS (170mM NaCl, 3.3 mM KCl, 1.8 mM Na2HPO4, 10.6 mM KH2PO4; pH 7.4) then 300µl of High Salt Lysis buffer or TNN buffer was added to cells on ice, then cells were scraped and samples collected in 1.5ml centrifuge tubes.

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Centrifuge tubes were then left rotating at 4oC for 20 minutes followed by centrifugation at 4oC for 20 minutes at 12,000 rpm. Supernatant were carefully transferred into fresh centrifuged tubes and pellets were discarded.

2.12.2 Determination of protein concentration

The Bradford protein assay was used for determining protein concentration from cell extracts. The Bradford’s assay is a colorimetric assay which is based on Bradford dye- binding methodto measure total protein concentration. The principle behind this assay is based on colour change of Coomassie brilliant blue G-250 dye after binding to different concentration of proteins. The concertation of protein samples then can be measured by measuring the absorbance at 595 nm.

The Bio-Rad working solution was prepared by adding 800 µl of distilled water to 200 µl of the Bio-Rad reagent (1 part of water to 4 part of the Bio-Rad reagent) in a 1.6 ml, 1 cm path visible cuvette. In order to calibrate 2 µl of distilled water was used and the spectrophotometer was set at 595 nm and was used as blank. To normalise the protein samples concentration 2 µl of samples was added to diluted dye and mixed and the absorbance of the solution was measured. Equal amounts of protein were mixed with 3 X SDS sample buffer (1M Tris pH 6.95 (Sigma, UK), 10% glycerol (Sigma, UK), 5% 2- Mercaptoethanol (Sigma, UK), 0.6g SDS (Sigma, UK)) and either loaded on SDS PAGE on the same day or left at -20oC.

2.12.3 SDS polyacrylamide gel electrophoresis

The first step in studying proteins by western blot is to separate them on SDS gel by means of the electrophoresis technique. SDS is an anionic detergent that binds to polypeptide chains of proteins in a manner that is proportional to molecular mass of the polypeptide. Smaller molecules move faster than the larger molecules on a poly acrylamide gel as it contains pores. In order to make sure that the charge to mass ratio is the same among the samples, protein samples were denatured incubating at 95oC for 5

85 min in SDS sample buffer (187mM Tris, 30% Glycerol, 6% SDS,15% 2-mercaptoethanol, 0.01% bromophenol blue).

To detect proteins SDS-PAGE was performed using 10% gels, components of 10% gel and volumes are shown in Table 2.4.

Table 2.4.The components and volumes required to create the resolving gel for one 10% acrylamide.

Component Volume

Deionised water 5.24 mL

Acrylamide (Fisher, UK) 4.67 mL

1.5 M Tris (pH8.95) (Fisher, UK) 3.5 mL

10% SDS (Fisher, UK) 0.14 mL

10% Ammonium persulphate (Sigma, UK) 0.075 mL

0.2 M EDTA(Fisher, UK) 0.14 mL

86 Table 2.5.The components and volumes required to create 6% stacking gel for one stacking gel.

Component Volume

Deionised water 3.37 mL

Acrylamide (Fisher, UK) 0.835 mL

1.5 M Tris (pH6.95) (Fisher, UK) 0.625 mL

10% SDS (Fisher, UK) 0.050 mL

10% Ammonium persulphate (Sigma, UK) 0.075 mL

0.2 M EDTA (Fisher, UK) 0.050 mL

TEMED (Sigma, UK) 0.0085 mL

According to manufacturer’s guide line the gel casting apparatus was set-up. The components in Table 2.4 were prepared and the mixture was poured between the plates. To prevent gel from drying and removing the air bubbles after the resolving gel was poured into the gel casting apparatus, 500 µl of distilled water was poured on top. Once the gel was set the water was removed stacking gel mixture was poured on top of resolving gel and 1.5 mm wide well combs were inserted into the stacking gel solution (Table 2.5). Since the stacking gel has lower percentage of acrylamide and lower pH comparing to resolving gel it allows the proteins to be stacked before they leave the stacking gel and entering resolving gel. When the stacking was set the gels were fitted into electrophoresis mini buffer tank and the tank was filled with 1x SDS-PAGE running (25 mM Tris, 190 mM glycine, 3 mM SDS). Protein samples were boiled at 95oC for 5 minutes and loaded into the wells of gel. 5 µl of prestained protein ladder (Fisher, UK) was loaded in every gel and the samples were run at 90V until they enter the resolving gel and then at 110V.

87 2.12.4 Western transfer

When proteins resolved on the gels they were transferred to a polyvinylidene fluoride PVDF membrane (Millipore, UK) using western transfer method. Western transfer apparatus (Bio-Rad, UK) was set up according to manufacturer’s guideline and the tank was filled with western transfer buffer (28 mM Tris, 0.15 M glycine, 20% methanol). The transfer was carried out for 90 minutes in 0.4amps with ice pack changed every 45 minutes. When the transfer was completed the PVDF membranes were blocked in 5% dried milk in 1x phosphate buffered saline (PBS) (170mM NaCl, 3.3 mM KCl, 1.8 mM Na2HPO4, 10.6 mM KH2PO4; pH 7.4) for 1 hour. The membrane then was incubated with desired antibody in 2.5% milk/0.1% PBS Tween (PBS + 0.1% Tween-20)overnight on a rocking platform at 4oC.

The day after incubation membranes were washed three times each time 10 minutes in PBS 0.1% tween, and then membrane was incubated in relevant secondary anti-rabbit or anti-mouse IgG horseradish peroxidise conjugated secondary antibody in 2.5% milk PBS/tween.

Three further washes with PBS Tween (each wash for 10 minutes) were carried out. In order to visualise the proteins SuperSignal™ West Femto (Thermo Fisher, UK) or SuperSignal™ West Pico (Thermo Fisher, UK) Chemiluminescent reagent was added on to the membrane and proteins were visualised on medical X-ray films (Fujifilm, UK) using developer.

2.12.5 Western blot quantification

Image J 1.48 was used to quantify western blot results with readings of each protein band normalized to their corresponding actin band. Readings were then expressed relative to the control lane and results were then presented as bar charts. As shown below in Figure 2.3 a square was drawn around the protein bands, and by selecting a specific tool protein bands were represented as separate peaks. The software in a different window then calculated the density represented by peaks.

88 Figure 2.3.Summary of ImageJ quantification.

2.12.6 Striping the membranes

In order to strip the antibody off the membranes after detecting the first protein whenever necessary, the membranes were incubated in striping buffer (100 mM 2-Mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) for 30 minutes at 50oC and washed three times in 0.1% PBS tween for 10 minutes. Membranes were then blocked with the desired primary antibody.