Chapter 2 Materials and Methods
2.3 Methods
2.3.11 Protein Analysis
Protein analysis was performed to analyse the expression of proteins and protein isoforms in patient samples through use of Western Immunoblotting.
2.3.11.1 Bradford Assay – Coomassie Dye Based Protein Assay
Coomassie dye is used to quantify extracted protein lysate. The dye binds to the extracted protein resulting in a shift from the dyes standard absorbance maximum at 465nm (reddish brown) to 595nm (blue). The binding efficiency of the Coomassie dye ligands bound to protein is proportional to the number of positive charges on the protein. Generally the protein must be at least 3kDa in size to be bound by the dye.
Bradford assays were set up in 96 well plates. BSA provided with the assay was diluted into a series of standards according to Table 2.9 used to quantify protein samples, all standards were set up in triplicate. 250µl of Coomassie dye was mixed with 5µl of protein or standard sample. Protein samples were set up in duplicate due to limited availability. The plate was analysed within an hour of preparation, absorbance was measured at 595nm using the Fluostar Omega plate reader.
Tube Volume of Diluent
Volume and Source of BSA Final BSA Concentration A 0 300µl of stock 2000µg/ml B 125µl 375µl of stock 1500µg/ml C 325µl 325µl of stock 1000µg/ml D 175µl 175µl of vial B dilution 750µg/ml E 325µl 325µl of vial C dilution 500µg/ml F 325µl 325µl of vial E dilution 250µg/ml
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G 325µl 325µl of vial F dilution 125µg/ml H 400µl 325µl of vial G dilution 25µg/ml
I 400µl 0 0µg/ml
Table 2.9. Dilution scheme for Bradford Assay standards.
2.3.11.2 Western Immunoblotting
Western immunoblotting was performed to detect the presence and quantity of selected protein. This was achieved by first separating proteins by weight and 3- D structure by using polyacrylamide gel electrophoresis, upon separation proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and probed by antibodies for the protein of interest.
Gel Electrophoresis
Amersham ECL Gel was briefly rinsed with dH2O and placed into an Amersham ECL Gel Box and then filled with 180ml 1x running buffer. The gel was run at 160V for 12 minutes, the gel comb was then removed and the wells filled with 6ml of 1x running buffer. Samples were then loaded into the gel with 10ul of Spectra Multicolor Broad Range Protein Ladder. Empty wells were filled with 30µl of urea buffer mixed with 5µl of loading buffer. The gel was run at 160V for 60 minutes.
Protein Transfer
The PVDF membrane was cut with clean scissors to match the size of gel, the membrane was activated by being soaked in 100% Methanol for one minute, after the incubation time the membrane was left submerged in 1x transfer buffer until needed.
Six pieces of filter paper matching the size of the gel and PVDF membrane were cut and soaked in 1x transfer buffer along with transfer sponges. Sponges were thoroughly squeezed and soaked in transfer buffer until all air bubbles had been removed.
After the gel had completed running it was detached from the plastic cassette and the stacking gel was removed. Keeping the gel moist, the PVDF membrane was carefully placed on the gel and sandwiched between the soaked filter paper and
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sponges (Figure 2.3). This stack was placed into a transfer cassette and a protein gel tank, making sure that the membrane faced the positive electrode. The tank was filled with 1x transfer buffer. The tank was either placed in a polystyrene box filled with ice or an ice pack was placed in the tank to maintain a low temperature during protein transfer. The samples were transferred by running the power pack at 100V for 60 minutes.
Figure 2.3. Western immunoblot set up for protein transfer to PVDF membrane adapted from abcam western blotting beginners guide.
Immunoblotting
After the run had finished, successful transfer was confirmed by viewing the transmission of the multi-coloured protein ladder to the PVDF membrane. The membrane was then blocked in 5% skimmed milk powder solution for at least 30 minutes at room temperature or overnight at 4ᵒC on a rocking platform. The membrane was washed three times with TBST solution and stored in TBST to prevent it from drying out. The membrane was then cut appropriately using the protein ladder as a guide to separate the membrane containing the loading control protein from the target protein. Antibodies were diluted as displayed in Section 2.2.9.2. Membrane sections were placed in separate 50ml falcon tubes with 4mls of 50% 5% skimmed milk solution (2.5% after dilution) and 50% TBST solution with the respective diluted antibody. The falcons containing the membranes and antibody mix was incubated at room temperature for one hour on a roller mixer, or for up to 18 hours at 4ᵒC. The membranes were then washed a further three times in TBST solution and the incubation process was repeated with the secondary antibody for one hour, followed by the same wash steps.
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Finally to visualise the proteins the PVDF membrane was stained with Amersham Enhanced Chemiluminescence Prime Western Blotting Detection Reagent. This was performed by mixing 500ul of reagent A and B and adding the mixture to the membrane, and leaving at room temperature for one minute. After the incubation period excess solution was carefully removed by tipping off the membrane onto tissue paper. The membrane was then placed into a plastic folder, air bubbles were carefully removed and the blot was inserted into the G:Box for imaging and analysed using ImageJ software (Version 1.48).
Protein Stripping
Some membranes were stripped from their original antibodies due to the necessity of re-probing with different target antibodies, or the need to visualise a stronger antibody signal. The selected PVDF membrane was washed with TBST solution and immersed in Restore™ Western Blot Stripping Buffer (Thermo Scientific) for one hour at room temperature in a shaker. After stripping the membrane was washed a further three times with TBST solution and blocked in 5% milk solution before being incubated with the desired primary antibody, following the standard western immunoblotting protocol (Section 2.3.7.1.3).
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