Western Blotting 76

Western blotting (immunoblotting) methods are based on antigen-antibody interactions. A known quantity of total protein is separated based on

molecular weight (Mw) using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Seperated proteins are then transferred from the gel to a membrane prior to probing of interest-specific antibody and a labelled secondary antibody. Fluorescent detection is employed in this thesis to visualise protein bands: the fluorophore attached to the conjugated secondary antibody is excited by specific wavelength light and a photo sensor such as charged coupled device (CCD) camera equipped with appropriate emission filters detects emission. The captured digital image of the western blot can then be used for quantitative analysis such as determination of protein band size.

2.5.1 Protein Extraction and Precleaning

A small piece of tissue (~30mg) was cut on dry ice and added to 2ml tubes containing magnetic beads and 200µl of Tris Lysis buffer [50mM Tris/HCl (pH 8.0), 1% (v/v) Triton X-100, 150mM NaCl, 5mM EDTA, complete protease inhibitor tablets (1ml of 0.1% of lysis buffer comprising of protenase phosphatase), which was followed by homogenizing the tissue on Tissue Lyser LT (Qiagen, Germany) for 2 minutes. Each sample was then transferred to a different 2ml tube and centrifuged at 4°C for 5 minutes at 10000g after which the protein supernatant was removed and stored.

The protein samples were precleaned using Sepharose conjugate (Rec- Protein Sepharose 4B Conjugate, Invitrogen Ltd, USA), washed in lysis buffer twice and then added to each sample at a 0.1v/v dilution. The samples were then rotated for 2 hours at 4°C followed by microcentrifugation at 4°C for 10 minutes at 10000g. Later, the supernatants were transferred to clean 2ml tube and stored at -80°C until further analysis.

2.5.2 Protein concentration measurement

Protein concentration was measured using Bradford dye-binding (Protein dye- BioRad) assay (Bradford 1976). The Coomassie Blue G-250 dye within the

Bradford reagent binds to the protein sample, which shifts the absorbance from 465nm to 595nm and this change in absorbance at 595nm is directly proportional to the amount of protein present in any given sample. Bradford reagent was prepared by adding 100mg of Comassie Brilliant Blue G-250 (Bio-Rad) to 50ml of 95% ethanol. To this, 100ml of 85% (w/v) phosphoric acid was added and the solution was placed on a stirrer for 30 min, then made up to a final volume of 1L by adding dH2O and filtered through

Whatman grade 1 filter paper. 5µl of protein samples were added to the wells of 96-well plates (Costar 96-well Polystyrene plate, NY, USA) followed by addition of 250µl of Bradford reagent and incubated on a plate shaker at 370C for 3 minutes. The microplate was then read at 595nm using microplate reader (Dynex Technologies, MRXII) and software Revelation V.4.25. The software extrapolated the concentrations of samples by providing a standard curve constructed from the different dilutions of bovine serum albumin (BSA) used as standards, ranging from 0-3mg/ml. Samples were diluted using dH2O

in order to fall within the linear range of the standard. Quality control was included in every assay to ensure assay precision. Inter- and intra assay CVs were <5% and <2%, respectively.

2.5.3 Preparation of Gels

1.5mm thick polyacrylamide gels were made in advance and stored at 40_{C for}

up to 2 days before use. Glass plates (Mini PROTEAN® _{System, BioRad}

Laboratories Ltd, USA) were used to make up resolving and stacking gel as outlined in Table 2.5, however, TEMED and APS were added just before casting the gels. The glass plates were assembled and the resolving gels poured carefully (making sure of no leakages and also keeping free from any air bubbles) up to the level 1cm below the comb teeth. Finally, 4% stacking gel was poured gently, avoiding air bubbles before the 10-well comb (1.5mm spacer) (Mini PROTEAN® Comb 10-well, BioRad Laboratories Ltd, USA) was inserted and incubated to polymerize for 30 min.

Reagent Resolving (12%) Stacking (4%) Acrylamide 4ml _670µl Tris-HCl 2.5ml (1.5M; pH 8.0) 2.4ml (0.5M; pH 6.8) 20% (w/v) SDS _{50µl 50µl} dH2O 3.4ml 3.075ml APS (10%w/v) _{50µl 50µl} TEMED _{15µl 15µl}

Table 2.5 Components of western blot resolving and stacking gels

used in SDS-PAGE.

2.5.4 Western Blot Protocol

All reagents used in this protocol were purchased from Sigma-Aldrich, USA, unless otherwise stated. All samples were diluted to 1µg/µl in lysis buffer, (0.1M Tris-HCl pH 6.8, 20% glycerol, 4% (w/v) SDS, 1% bromophenol blue and 3% β-mercaptoethanol) (final volume of 100µl). Protein samples were denatured by heating at 700_{C for 5 minutes and were loaded (10/20µg per}

well depending on the protein of interest) with the addition of a full-range pre- stained protein molecular weight marker (PageRuler™Plus, Thermo Scientific, USA; Product No. 26619; Lot No. 00112289) into one well per gel and electrophoresed at 200V for 60 minutes. Sponges, blotting paper (Gel Blotting Paper Gb002; Scientific laboratories Ltd, Nottingham, UK), and nitrocellulose membrane (Amersham™ Hybond ECL, 0.45µm thick, GE healthcare Life Sciences, UK) were cut to size and soaked in transfer buffer before use. The gel was carefully separated from the glass plates by making use of transfer buffer (pH 8.0) and was ‘sandwiched’ between sponges (x2), filter paper (x2) and nitrocellulose membrane (x1) as shown in Figure 2.3. The cassettes were placed between electrodes in the gel tank and incubated with an ice block to prevent the apparatus from over heating and finally the blot transfer was run at 25V for 2 hours. Transfer was confirmed visually by placing the blot in 0.2% Ponceau S staining solution. Later the blots were blocked in 5% BSA in TBST

for 1 hour at room temperature. Blots were then incubated overnight at 40C with the primary antibody of interest (diluted in 5% TBST).

Blots were washed extensively 4 times for 5 minutes each using wash buffer (TBST) and then incubated in darkness for 2 hours with fluorescent secondary antibody specific to the species of the primary antibody. Blots were again washed using TBST every 5 min (x4) before visualisation. The blots were scanned under the Odyssey® Infrared Imaging System (LI-COR Biosciences, Cambridge, UK) using appropriate filters (700/800nm channel) and finally proteins bands were visualised and quantified using the software Image Studio Lite™ v.2.0. The size of the visualised protein band was confirmed with the position of the molecular weight marker.

Figure 2.3 Gel assembly set up utilised during transfer process for western

blotting.

Contents Running Buffer Transfer Buffer Wash Buffer

(TBS)

Tris 0.025M 0.025M 20mM

Glycine 0.192M 0.192M _

SDS 0.1% _ _

NaCl _ _ 0.15mM

Table 2.6 Components of buffers utilised in western blotting