WESTERN BLOTTING

2.4.1 DETERMINATION OF PROTEIN CONCENTRATION

Protein concentrations were determined calorimetrically using the BCA (bicinchoninic acid) protein assay kit.

2.4.2 MAKING: SDS-PAGE gel

Using Bio-Rad mini Protean II Gel system 8 % or 10 % SDS-PAGE gel was made up, respectively, according to manufacturer’s protocol. First the resolving gel was prepared by adding 30 % Acrylamide: Bisacrylamide (37.5:1),1.5M Tris-HCL tris (hydroxymethyl) amino-methane, pH adjusted with HCl], pH 8.0), pH 8.8, 10% SDS, deionised water, 10% ammonium persulfate (APS) and N, N, N’, N’- Tetramethylethylenediamine (TEMED). The mixture was poured into the casting system to gain the length of gel required, leaving sufficient space for the stacking gel and then allowed to

set at 20 o_{C for 30 minutes. Apart from 1.5 M Tris-HCl, pH 8.8 (substituted}

by 0.5 M Tris-HCl, pH 6.8), the stacking gel was prepared using all of the reagents as described above. The mixture was subsequently applied on top of the pre-set resolving gel and a comb was placed to allow Well

formation. The gel was allowed to set at 20 o_{C for 20 minutes. The gel was}

located into the electrophoresis running system and adequate running

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2.4.3 WESTERN BLOTTING OF CELL LYSATES AND CONDITIONED MEDIA

Sample preparation

Western blotting was used to assess protein levels in cell lysates and conditioned media from both treated and untreated prostate cancer cell lines. Protein lysates were prepared by adding equal amounts of radio- immunoprecipitation lysis buffer 1x RIPA buffer to disrupt cells & tissues, centrifuged at 8,000 rpm for 5 minutes and resulting protein lysates were quantified using a BCA (bicinchoninic acid) protein assay kit. 5 µl of each cell supernatant or standard was added to 96 well plate and a 195 µl of a mixture of CuSO4, PBS and BCA solution, was added to each well (4 µl CuSO4, 5 µl PBS and 200 µl of BCA solution) each well or sample.

Followed by incubation for 20-30 minutes at 37°Cand the absorbance was

measured using a plate reader (Multiskan Ascent 96/384 Plate Reader) at 570 nm. Samples were adjusted to equal amounts using standard 2X Laemmli buffer and placed in a boiling water bath for 5 min, and then allowed to cool at room temperature. The proteins were separated by SDS- PAGE and transferred to poly vinylidene difluoride (PVDF) membranes at 100 V for one hour in a transfer buffer containing 20 mM tris, 150 mM glycine, and 20 % methanol. PVDF membranes were blocked in tris buffered saline (TBS) containing 0.1 % TIen-20 and 5 % BSA for one or two hours. The PVDF membranes were incubated with the relevant

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primary antibodies overnight at 4°C. On the following day, membranes

were washed thoroughly four times in 60min with TBS-0.1% Tween before incubation with the appropriate secondary antibody for one hour at room

temperature. Antibody complexes were visualized using

chemiluminescence ECL+_{. The appropriate positive and negative controls}

were utilised. The densities were measured using a scanning densitometer coupled to scanning software ImageQuant™.

Table 2.2 Table demonstrating antibodies used for western blotting with

concentrations and relevant secondary antibodies.

Primary antibody Concentration Secondary

antibody

Concentra tion

Phospho – AKT 1: 1000 Anti – rabbit 1: 5000 Phospho –QRFP 1: 4000 Anti – rabbit 1: 12000 Phospho – GPR103 1 :3000 Anti – rabbit 1: 8000 Phospho – P38 1: 1000 Anti – rabbit 1: 2000 Phospho – JNK 1 :1000 Anti – rabbit 1: 2000 Phospho – AGR2 1: 1000 Anti – sheep 1: 4000 Phospho–

Csapease-3

1: 1000 Anti – rabbit 1: 1000

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2.4.4 MAPK- (ERK1/2, p38 and JNK1/2 MAPK) AND AKT ACTIVATION ANALYSIS

Prostate cancer cell line PC3 and DU145 cells were serum starved overnight seeded on to 6 well plates., and then treated with or without QRFP 100 nM for different time pointe (0 = Basal, 5, 15, 30 and 60 minutes) for MAPK ERK, P38, JNK and AKT studies and incubation with QRFP for 24 hours to detect downstream signalling pathways. Cells were then lysed with RIPA buffer and adjusted to equal amounts using Laemmli buffer, mixed, sonicated, boiled, centrifuged (5,000 rpm for 3 minutes), and stored at -20 ºC until use. 15-25 µg total protein per well was loaded onto 10 % SDS-PAGE gel. Following this, proteins were transferred onto PVDF membranes, blocked in tris buffered saline (TBS) containing 0.1% TIen-20 and 5 % BSA for one hour. The PVDF membranes were incubated with primary antibody for phosphorylated proteins (ERK/ p38/ JNKs/ Akt) overnight at 4°C respectively (table 2.2). Membranes were washed thoroughly for 60min with TBS-0.1% Tween before incubation with the secondary antibody for one hour at room temperature. Antibody complexes were visualized using chemiluminescence ECL prime. The densities were measured using a scanning densitometer coupled to scanning software Image Quant™.

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2.4.5 STRIPOING AND RE-PROBING MEMBRANES WITH TOTAL ANTIBODIES

DENSITOMETRY ANALYSES OF WESTERN BLOTS

The membranes were submerged in stripping buffer 10 % SDS (1M Tris- HCl, pH 6.8, β-mercaptoethanol) and incubated at 50oC for 30 minutes with occasional gentle agitation followed by washing with TBS-0.1 % Tween at room temperature 10 minutes twice. PVDF membranes were blocked in tris buffered saline (TBS) containing 0.1 % TIen-20 and 5% BSA for one hour at room temperature. The PVDF membranes were re-probed with primary antibody for total ERK, p38, JNK, Akt and GAPDH 1:1000 dilution overnight at 4°C. On the following day, membranes were washed thoroughly for 60min with TBS-0.1 % Tween before incubation with the secondary antibody for one hour at room temperature. Antibody complexes were visualized using chemiluminescence ECL prime. The densities were measured using a scanning densitometer coupled to scanning software Image Quant™.