Materials & Methods
Chapter 2 Materials and methods
b lo ck set at 100 °C fo r 10 m in to denature the proteins. N e x t, a ra in b o w p ro te in m arker and the p ro te in sam ples w ere loaded in to the w e lls o f the lo a d in g gel and the e le ctrop h oresis c e ll was subjected to 2 00 V fo r around 45 m in o r u n til the p ro te in sam ples had reached the b otto m o f the gel.
2.2.4.4 Transfer o f proteins
F o llo w in g separation o f the p ro te in sam ples the gels w ere rem oved fro m the glass plates in w h ic h the y w ere set and assem bled in p la s tic tra n sfe r cassettes as show n in fig u re 2 .1 , to m ake a tra n sfe r ‘ sa nd w ich ’ .
Nitrocellulose membrane Gel Sponge Paper Paper Sponge Figure 2.1 Diagram to show the assembly o f the blotting sandwich
The sandw ich was assem bled such th a t the gel was nearer to the b la c k side o f the cassette (cathode) to ensure th a t the p ro te in s tra v e lle d fro m the gel to the n itro c e llu lo s e m em brane, w h ic h was at the anode side o f the cassette. I t was then placed in the e le ctrop h oresis c e ll. A n ice pack was in clu d e d to ensure the electrophoresis c e ll d id n o t overheat. The c e ll was fille d w ith tra n sfe r b u ffe r (see appendix 1) and a m a gnetic s tirre r was added to guarantee adequate c irc u la tio n o f tra n sfe r b u ffe r and to m a in ta in a constant tem perature. The
Chapter 2 Materials and methods
electrophoresis cell was then subjected to 100 V for 1 h to transfer the protein from the gel to the nitrocellulose membrane.
The membrane was then incubated with Ponceau S solution to ensure transfer success, which was subsequently washed off with TBS-Tween washes (see appendix 1 for recipes).
2.2.4.S Application of primary and secondary antibodies
Subject to transfer success, the membrane was then blocked with a 5 % w/v dried milk solution made up in TBS-Tween for 1 h. Whilst blocking, the membrane and block were rocked gently on a Stuart rocker (Bibby Scientific Ltd, Staffordshire, UK). After blocking, the membrane was incubated with primary antibody (see table 2.6) overnight at 4 °C on a Stuart roller (Bibby Scientific Ltd, Staffordshire, UK).
Chapter 2 Materials and methods
Protein detected 1* antibody concentration 2° antibody concentration
p-MAPK CST p-MAPk polyclonal at
1/1000 in 1% w/v dried milk in TBS-Tween with 0.05% SA overnight at 4°C
EnVision rabbit HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
p-Akt CST p-Akt polyclonal at
1/1000 in 1% w/v dried milk in TBS-Tween with 0.05% SA overnight at 4°C
EnVision rabbit HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
p-ER 118 NEB p-ER 118 monoclonal
antibody at 1/1000 in 0.05% POD v/v in TBS-Tween overnight at 4°C
EnVision rabbit HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
p-ER 167 CST p-ER 167 antibody at
1/1000 in 1% dried milk w/v in TBS-Tween overnight at 4°C
EnVision rabbit HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
Total-ER DAKO total ER clone ID5
mouse antibody at 1/1000 in 0.05% v/v POD in TBS-Tween overnight at 4°C
EnVision mouse HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
p-EGFR Biosource pEGFR 1068 rabbit
primary antibody at 1/1000 in 1% w/v dried milk in TBS- Tween with 0.05% SA overnight at 4°C
EnVision rabbit HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
Chapter 2 Materials and methods
Total-EGFR NeoM arkers EGFR mouse
monoclonal antibody at 1/1000 in 1% w/v dried milk in TBS- Tween with 0.05% SA overnight at 4°C
EnVision mouse HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
Total COX-2 NEB total COX2 rabbit
polyclonal prim ary antibody 1/1000 in 1% w/v dried milk in TBS-Tween with 0.05% sodium azide overnight at 4°C
EnVision rabbit HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
Total Src CST total Src mouse
polyclonal primary antibody at 1/1000 in 1% w/v dried milk in TBS-Tween with 0.05% SA overnight at 4°C.
EnVision mouse HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
p-Src CST p-Src Tyr 414 rabbit
polyclonal primary antibody at 1/1000 in 1% w/v dried milk in TBS-Tween with 0.05% SA overnight at 4°C.
EnVision rabbit HRP labelled polymer at 1/100000 in 1% w/v dried milk in TBS-Tween
T able 2.6 Table to show the concentrations o f l°and 2° antibody for each protein detected.
After sufficient incubation with the primary antibody, the membrane was rocked gently for 3 x 5 min washes in TBS-Tween. The membrane was incubated in the presence of the secondary antibody (as shown in table 2.6) for 75 min.
Chapter 2 Materials and methods
2.2.4.6 Visualisation of proteins by chemiluminescence
The membranes were washed three times in TBS-Tween for 10 min. The membranes were then placed into a clean plastic folder and were treated with luminol/peroxide based enhanced chemiluminescence (ECL) reagent (Pico, Dura or Femto). The luminol present within this reagent is oxidised by horseradish peroxidase to produce an excited state product, which emits photons of light as it decays to a lower energy state. Kodak autoradiography film was then placed over the membranes and the film was developed using a X-O-graph Compact X2 X-ray developer (X-O-graph Imaging System, Tetbury, UK) in a dark room.
2.2.5 Statistical Analysis
All statistics throughout this thesis were carried out on Minitab for Windows. Confidence intervals were set at 95% and significance was defined as p=<0.05.
Chapter 3 Transcutaneous delivery of signal transduction inhibitors and tamoxifen