Cells were seeded in T25 flasks and incubated for 24h at 37°C and 5% CO2, and the following day cells were exposed to the desired drugs and functionalised NDs for 48h at 37°C and 5% CO2 (the time point 48h was chosen as the optimum time point to see the effect of the nanocomplexes at the protein level). Cells were harvested in cold PBS and centrifuged at 400 g for 10min at 4°C, the supernatant was discarded, and the pellet was suspended in 200ul of RIPA buffer with 0.1% of protease inhibitor and kept on ice for 15 min. The suspension was centrifuged at 400g for 15 min at 4°C, the supernatant was transferred to another tube and kept at – 80°C.
Protein Concentration Measurement (Bradford assay)
1. Serial concentrations were performed from the protein standard (BSA) (1500, 750, 375, 188, 94, and H2O).
2. 5ul of protein samples were added in duplicate in a 96 well plate.
3. 250ul of Bradford reagent was added to each well, and the plate was incubated on a shaker at RT for 15 min.
4. The plate was read at 595nm using microplate reader.
5. Linear regression and interpolation of protein concentration from the standard curve was performed using GraphPad prism 5 software.
6. The protein samples were normalised to load equal amount of protein concentration of each sample into each well.
Western Blotting
Western blotting is a membrane immunoblotting technique used to detect the presence of specific target proteins. Following SDS-PAGE proteins were electrophoretically transferred to a thin membrane composed of nitrocellulose or PVDF (polyvinylidene difluoride). The membrane was placed on top of the gel, overlaid with blotting paper or sponges, this stack or transfer sandwich was then loaded into an electrophoretic apparatus. The transfer sandwich was immersed in buffer and an electric current was applied. This applied electric current will cause separated proteins contained within the gel to migrate and bind to the membrane. The membrane then was probed with specific
57 antibodies for target proteins of interest. Individual protein bands can be visualised using a HRP labelled secondary antibody and a suitable chemiluminescence reagent.
Sample Preparation
1. Protein samples were thawed on ice for 30 minutes.
2. Protein samples were diluted to 30ug in a total volume of 30ul with RIPA buffer in fresh Eppendorf tubes.
3. Loading dye was added to each tube in 1:5 ratio of the protein sample. 4. 4ul of loading dye was added to each sample, then all protein samples were
kept in boiled water for 30 secs and transferred onto ice for 3 mins. 5. 5ul of a loading protein marker was loaded in the first well.
6. 30ul of protein samples were loaded into each well.
7. The rig was connected to the power supply and run at 120v for 60 mins.
Coomassie Blue Staining
1. Gel was removed from the transfer sandwich and placed in a small container filled with deionised water and briefly rinsed.
2. Gel was covered with Coomassie blue and placed on an orbital shaker for 30min.
3. Coomassie stain was poured off and gel was rinsed with deionised water. 4. The gel was covered with the de-stain solution (40% methanol and 10% glacial
acetic acid) for 10mins.
Western Blot
1.Transfer buffer was kept at 40C prior to use.
2.Blotting paper and membrane were cut to the required size. 3.Blotting paper was immersed in transfer buffer.
4.The PVDF membrane was immersed in 70% methanol for 1 minute to be activated and then immersed in transfer buffer and allowed to equilibrate for at least 15 mins.
5.The top and bottom portions of the gel were cut, and the top right-hand corner was removed for orientation.
58 6. The gel was carefully removed from the gel cassette and placed in a tray of
deionised water.
7. The transfer sandwich was prepared as outlined:
a. Two pieces of filter paper soaked with transfer buffer. b. PVDF Membrane.
c. The gel.
d. Two pieces of filter paper soaked with transfer buffer.
8. Air bubbles were removed by rolling the surface of the sandwich.
9. The rig was connected to the power supply and run at 32 m/Am for 1 hour to transfer protein samples.
Protein Detection
1. The transfer sandwich was de-assembled.
2. The membrane was carefully peeled away from the gel and the side which was in contact with the gel was facing upwards. The notched corner should now be on the left-hand side.
3. Next the membrane was immersed in appropriate blocking solution such as 5% w/v Marvel milk protein and incubated at room temperature for 1 hour on an orbital shaker at 50revs/min to block non-specific binding sites. 4. The membrane was immersed in 10mls of diluted primary antibody
(1:1000 of Caspase 3, or 1:1000 of P-gp), then incubated at 40C overnight on an orbital shaker.
5. Following overnight incubation with the primary antibody, the membrane was rinsed quickly with 1X TBST to remove most of the residual unbound antibody, then three washes were performed (1 x 15min and 2 x 5min washes in 1x TBST) on an orbital shaker.
6. Afterwards, the membrane was transferred to a new tube containing the suitable secondary antibody (1:1000 of Goat Anti-Mouse Immunoglobulins HRP) and incubated on an orbital shaker at room temperature for 1 hour, then three washes were performed (1 x 15min and 2 x 5min washes in 1x TBST) on an orbital shaker.
59
Developing Blots
1. Luminol detection reagent was prepared by adding an equal volume of detection solution A (Luminol enhancer solution) with an equal volume of detection solution B (Peroxide solution) in a tinfoil covered tube and then mixed thoroughly.
2. The membrane was placed between two plastic strips and the excess moisture was removed by pressing down with a piece of tissue paper. 3. The membrane was placed within the plastic sheet inside the LAS-4000
luminescent Image analyser.
4. The chemiluminescence option was selected and the images were taken at appropriate times.
5. The black and white balance was adjusted to obtain the optimum image, then the image was saved.
Stripping blots
1. Blots were stripped in order to examine expression of multiple proteins. 2. The membrane was activated using 70% ethanol for 5 mins at RT.
3. The membrane was placed in a 50 ml tube and the stripping buffer was added as following:
a. 3.12ml of Tris-HCL (62.5mM, pH 6.8). b. 5ml of 20% SDS.
c. 0.415ul of β-mercaptoethanol. d. The volume was made up to 50 ml.
4. The tube was incubated in a water bath for 45 mins at 50 0C. 5. Three washes were performed for 5 mins each with TBST. 6. The membrane was blocked with 5% milk for 1 hour at RT.
7. The new primary antibody (ß-Actin) was applied and incubated for two hours at RT.
60
2.6 Tissue culture (Ex vivo)