Protein Analysis

2.2.4.1 Protein Extraction

Whole cell protein extracts were obtained by direct lysis in RIPA buffer. RIPA buffer

was supplemented with cOmplete EDTA-free protease inhibitor. Media was aspirated

from cells and cells washed with PBS. 60μl RIPA solution was added per well (in a 6 well plate, scaled volumes were applied for other plasticware). Cells were scraped

using a silicon scraper and collected in microcentrifuge tubes. Samples were

centrifuged at 12,000 x g for 15 minutes at 4°C and supernatant containing protein

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2.2.4.2 Assessment of Protein Concentration

Protein concentration was determined via Bradford assay. Bradford assay reagent

contains a Coommassie dye which exhibits an absorbance shift when bound to

specific amino acid residues in proteins, observable by a colour shift from red to blue.

Stock Bovine Serum Albumin (BSA) was diluted to a concentration range of 0, 0.5,

1.0, 1.5, 2.0, 2.5, 3.0 and 3.5μg/well in a 96 well plate format. 20μl of Biorad protein dye was pipetted into each well for the protein standard and sample wells in triplicate.

The protein samples to be quantified were diluted 1:400 in distilled water and added

to each well. Plates were loaded onto the Multiskan Ascent plate reader and

absorbance measured at 595nm. Protein concentrations of the samples were

calculated by reference to known standards.

2.2.4.3 SDS-PAGE

Appropriate concentrations of protein were diluted in Laemmli buffer and heated to

100˚C for 5 minutes and quickly cooled on ice. Proteins were resolved on discontinuous polyacrylamide gels using the Invitrogen XCell SureLock Mini-cell

apparatus. Gels were prepared in disposable plastic cassettes from two solutions to

form an upper stacking gel (usually 5%) and a lower resolving gel (variable %

depending on protein size). The stacking gel was prepared to pH 6.8 and the resolving

gel to pH 8.8. Polymerisation in the gel was instigated by the addition of TEMED and

10% APS. The resolving gel was poured into the cassette, overlayed with 100%

isopropanol and left to polymerise. Once set, isopropanol was rinsed off with distilled

water and the stacking gel overlayed and combs inserted.

Gels were inserted into the electrophoresis tank with running buffer. Equal

63 molecular weight marker. A constant voltage of 100V was applied until separation

had occurred. The cassette was opened and gel removed for Western blotting.

2.2.4.4 Western Blotting

Protein samples resolved by SDS-Page were transferred to PVDF membrane for

immunoprobing using a wet-blot method. Gel/membrane sandwiches were made

consisting of pre-soaked blotting pads on the cathode shell, followed by a pre-soaked

Whatman filter paper. The gel and the 100% methanol activated PVDF membrane

were orientated next, followed by another filter paper and finally two pre-soaked

blotting pads towards the anode shell. Once assembled the sandwich was rolled to

eliminate any bubbles and placed in the transfer tank with transfer buffer enriched

with variable volumes of methanol dependent upon protein size. Transfer was

performed at a constant voltage of 30V for 2 hour. Following transfer, the PVDF

membrane was air dried, reactivated in 100% methanol, and blocked in 5% w/v milk

powder in TBS-Tween for 1 hour. The membrane was subsequently incubated with

the primary antibody diluted in 5% milk TBS-Tween overnight at 4˚C. The membrane was washed 5 x in TBS-Tween, and subsequently incubated in the secondary

antibody conjugated to horseradish peroxidise in the appropriate species diluted in

5% milk TBS-Tween for 1 hour at room temperature. Following three washes in TBS-

Tween, the membrane was washed finally in distilled water. Chemiluminescent

signals were visualized by using ECL Plus Western Blotting Detection System either

onto autoradiography film or using a G:Box Chemie XX6.

2.2.4.5 Phospho-MAPK Array

Relative phosphorylation of 26 phospho-kinases was determined by Proteome

64 manufacturer’s specifications using 250μg total protein lysates. Briefly, cell lysates are diluted and mixed with a cocktail of biotinylated detection antibodies. The lysates

are then incubated overnight with the phosphor-MAKP array blot. The membrane was

washed several times to remove any unbound material. Streptavidin-HRP and

chemiluminsence detection reagents were applied producing a signal at each capture

spot corresponding to the amount of phosphorylated protein bound. Densitometry

was performed with individual phospho-proteins expressed as a percentage of

reference dots.

2.2.4.6 Enzyme-linked Immunosorbent Assay (ELISA)

Several ELISA kits were used throughout the study for detection of PRIP1, sST2, PRL

and IGFBP1. For detection of PRIP1, total protein lysates were used, for the detection

of sST2, PRL and IGFBP1, supernatant from cell culture was used. All ELISA kits

used were solid phase sandwich ELISAs.

In brief, a serial dilution of known protein concentration was added to an antibody

specific pre-coated microplate along with unknown samples. The plate was sealed

and incubated for 2 hours at 37˚C. Following incubation, the samples are aspirated and biotinylated detection antibody was added to each well and again sealed and

incubated for 1 hour at 37˚C. Following three washes, horseradish peroxidase (HRP) conjugated streptavidin was added to each well and incubated for 1 hour at 37˚C. Again, following three wash steps, a substrate solution was added to the wells and

colour develops in proportion to the amount of protein bound in the initial step. The

colour development was stopped and the intensity of the colour measured

immediately using a PheraStar microplate reader at 450nm with correction deducted

from 540nm. Results were derived using a 4-paramenter logistic regression analysis

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