Laura J. Sampson and Caroline Dart
7.3. Methods
7.3.1. Preparation of Arterial Homogenate:
Detergent-Based Method
3. Homogenise the powdered tissue in 2 mL of ice cold MBS (25-mM MES and 150-mM NaCl, pH 6.5) containing 1%
Triton-X (see Note 1) on ice with a hand-held homogeniser, then incubate on ice for a further 10 min.
4. Make the homogenate up to 45% sucrose by the addition of an equal volume of ice cold 90% sucrose in MBS and load the resulting 4 mL into a thin-walled ultracentrifuge tube. This forms the bottom layer of the discontinuous sucrose density gradient.
5. Overlay the sample with 4 mL of ice cold 35% sucrose pre-pared in MBS and then 4 mL of ice cold 5% sucrose again prepared in MBS.
6. Separate the buoyant and non-buoyant membrane fractions by centrifugation of the sucrose gradient at ∼260,000×g for 18 h at 4°C using an ultracentrifuge equipped with swing-out rotor (for example Sorvall TH-641). Remember to pre-cool the centrifuge before use and keep the gradients on ice until transferred to the centrifuge.
Fig. 7.1. Separation of caveolar and non-caveolar membrane fractions from rat aortic smooth muscle. (a) Relative choles-terol levels in each of ten 1-ml fractions collected from the top to the bottom of a discontinuous sucrose density gradient (shown schematically on left). (b) Western blot analysis of the fractions shown in A above to determine the localisation of the caveolae membrane marker, caveolin-1 (top panel), the non-caveolar, clathrin-coated pit protein, β-adaptin (middle panel) and the pore-forming subunit of the vascular KATP channel, Kir6.1 (adapted from ref. 18).
7. By carefully placing a pipette tip just under the surface at the very top of the gradient, collect consecutive 1-mL fractions and place these in pre-labelled tubes on ice. If the centrifuge tube is held up to light, the buoyant, cholesterol-enriched membranes are sometimes visible as a milky, light-scattering band at the 5–35% sucrose interface. Store the fractions at
−20°C until required for SDS-PAGE.
1. Place 2–3 aortae into a pestle that has previously been stored at −80°C (see Note 3). Pour a small quantity of liquid nitro-gen over the aortae and crush with the mortar until the tissue is reduced to a powder.
2. Homogenise the powdered tissue in 2 mL of 500-mM Na2CO3 pH 11 on ice with a hand-held homogeniser.
3. Disrupt the cell membranes by sonicating the homogenate (3
× 20 s bursts) on ice using a probe sonicator.
4. Make the homogenate up to 45% sucrose by the addition of an equal volume of ice cold 90% sucrose in MBS (25-mM MES and 150-mM NaCl, pH 6.5) and load the resulting 4 mL into a thin-walled ultracentrifuge tube. This forms the bottom layer of the discontinuous sucrose density gradient.
5. Overlay the sample with 4 mL of 35% sucrose prepared in MBS with 250-mM sodium carbonate and then 4 mL of 5% sucrose again prepared in MBS with 250-mM sodium carbonate.
6. Separate the buoyant and the non-buoyant membrane frac-tions by centrifugation of the sucrose gradient at ~260,000×g for 18 h at 4°C using an ultracentrifuge equipped with swing-out rotor (for example Sorvall TH-641).
7. By carefully placing a pipette tip just under the surface at the very top of the gradient, collect consecutive 1-mL fractions and place these in pre-labelled tubes on ice. If the centrifuge tube is held up to light, the buoyant, cholesterol-enriched membranes are sometimes visible as a milky, light-scattering band at the 5–35% sucrose interface. Store the fractions at
−20°C until required for SDS-PAGE.
1. These instructions assume the use of a Mini-PROTEAN 3 electrophoresis system (Bio-Rad).
2. Clean the glass plates with a 70% ethanol solution and dry before use.
3. Prepare a 0.75-mm thick, 10% resolving gel by mixing 2.5 mL of 1× resolving buffer with 3.33 mL of acrylamide/bis, 100 µL of 10% SDS and 3.97 mL of distilled water. To polymerise the gel add 100 µL of 10% ammonium persulphate solution and 5 µL of TEMED. Pour the gel up to approximately 3 cm from the top to leave space for the stacking gel and overlay with 7.3.2. Preparation of
Arterial Homogenate:
Non-Detergent-Based Method
7.3.3. Polyacrylamide Gel Electrophoresis
isopropanol to prevent the top of the gel from drying out. The gel should polymerise in about 10–20 min depending on room temperature. (Keeping some of the gel mix in a universal tube is often a good idea to gauge the degree of polymerisation.) 4. Once the gel has set, pour off the isopropanol and rinse
the surface of the gel well with distilled water. Remove any excess water by inserting a strip of filter paper between the plates taking care not to disturb the surface of the gel.
5. Prepare the stacking gel by mixing 2.5 mL of 1× stack-ing buffer with 1.3 mL of acrylamide/bis, 100 µL of 10%
SDS, 6.0 mL of distilled water. To polymerise the gel add 100 µL of 10% ammonium persulphate solution and 10 µL of TEMED. Pour on top of the polymerised resolving gel to the top of the plates and insert the comb. The stacking gel should set within approximately 10 min depending upon the room temperature.
6. Prepare the running buffer by mixing 100 mL of 10× run-ning buffer with 900 mL of distilled water.
7. Once the stacking gel has set, carefully remove the comb and wash several times with distilled water.
8. Assemble the gel into the running module, and clip the module into the running tank. Fill the central reservoir with 1× running buffer.
9. Load up to 30 µL (depending on the comb/well size) of each fraction from the sucrose gradient in consecutive lanes reserving the first and last lanes for pre-stained markers. It is often useful to load different markers at each end to deline-ate samples and to easily identify the orientation of gel.
10. Fill the outer reservoir with 1× running buffer and connect to the power supply. Run the gel for about 60–90 min at 100 V (~20–30 mA). The position of the pre-stained mark-ers will give an indication of how far a protein of a particular molecular weight has migrated. The marker protein caveolin has a molecular weight of ~20 kDa. Therefore, ensure that the gel does not run for so long that these small proteins migrate off the bottom.
1. Proteins that have been separated by SDS-PAGE are trans-ferred to nitrocellulose membranes electrophoretically.
These instructions assume the use of a Mini-PROTEAN 3 electrophoresis system (Bio-Rad) and wet transfer.
2. Prepare 1.5 L of transfer buffer and place in cold room/
fridge to chill.
3. Cut four pieces of heavy weight blotting paper to the size of the fibre pads and place in a small volume of transfer volume to soak.
7.3.4. Western Blotting
4. Cut the nitrocellulose membrane to the size of the resolving gel, wet with distilled water and then place along with filter paper to soak in the transfer buffer.
5. Disconnect the gel unit from the power supply and disas-semble. Separate the glass plates and remove and discard the stacking gel.
6. Place one of the fibre pads onto a flat surface, lay two pieces of blotting paper on top of the pad and then place the gel carefully on top. Overlay the gel with the nitrocellulose membrane and roll over the surface of the membrane with something cylindrical (we typically use a plastic disposable stripette) to remove any bubbles and ensure good contact between the gel and membrane. Complete the sandwich by placing the remaining two pieces of blotting paper on top of the gel and overlay with the second fibre pad.
7. Clip into the gel holder cassette and insert into the transfer cell ensuring that the nitrocellulose membrane is between the gel and the anode. Place the transfer cell in the running tank and fill the tank with chilled transfer buffer. Surround the running tank with ice (or place the whole assembly in a cold room) and connect to the power supply.
8. Transfer proteins electrophoretically onto the nitrocellulose membrane at 100 mV (~300 mA) for approximately 1 h.
9. Disassemble the apparatus, carefully remove the membranes and rinse them briefly in TBS-T.
10. Block the membranes overnight at 4°C in blocking buffer.
11. Dilute the primary antibodies: adaptin (1:1,000) anti-caveolin 1 (1:1,000), anti-flotillin 1 (1:250), anti-flotillin 2 (1:5,000), anti-Kir6.1 (1:500) in primary antibody dilution buffer and incubate with the membranes for 1–2 h at room temperature.
12. Wash the membranes for 3 × 10 min in TBS-T solution then incubate with horseradish peroxidase-conjugated secondary antibodies (1:20,000 dilution in secondary antibody dilu-tion buffer) for a further hour at room temperature.
13. Visualise the labelled bands using enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotechnologies) and exposure to light-sensitive film (Hyperfilm™ ECL™; Amersham).
1. Lipid raft regions are only resistant to solubilisation by cold detergents. It is therefore imperative that the detergent-based