Acknowledgments
6.3. Methods
6.3.1. Examining Endocytosis by Biotinylation
biotin). Each flask requires two, 20-min washes with glutath-ione buffer at 4°C.
8. Remove the glutathione buffer and quench residual glutathione with 2 ml of iodoacetamide solution for 5 min at 4°C.
9. Remove the iodoacetamide buffer and lyse the cells in 1-ml of extraction buffer at 4°C with gentle agitation. Lysis is complete when cell clumps are no longer visible to the eye.
10. Transfer each lysate to a 1.5-ml microcentrifuge tube and cen-trifuge at 15,000 × g in a cooled (4°C) bench top microcentri-fuge for 90 min. This clears the lysate of any residual cellular debris (see Note 5).
11. Determine the protein content of each lysate using a cell lysate total protein assay (see Subheading 6.3.3).
12. Dilute the lysates with extraction buffer so that they are all of the same concentration. The lysates should then be stored on ice.
13. Mix equal volumes of each lysate (containing equal quantities of total protein) with 50-µl of neutravidin beads in microcen-trifuge tubes and incubate them with rotation at 4°C for 2 h.
This allows the biotinylated proteins in the lysate to bind to the neutravidin beads.
14. Pellet the neutravidin beads by centrifugation and remove the supernatant (see Note 6).
15. Wash the neutravidin bead pellet four times with extraction buffer to remove any non-specifically bound proteins.
16. Elute the biotinylated proteins from the neutravidin beads by re-suspension of the washed neutravidin bead pellet in 50-µl of reduc-ing sample buffer and incubate for 15 min at room temperature.
17. Pellet the neutravidin beads by centrifugation and resolve the eluted proteins contained within the reducing sample buffer by SDS-PAGE (see Subheading 6.3.4).
1. Culture cells expressing KATP-HA to confluence in 25-cm2 culture flasks. One flask is required for each time-point to be investigated (see Note 4).
2. Remove the culture medium and rinse four times in 1× PBS.
3. Biotinylate cell surface channels with 2 ml per flask of chilled (4°C) biotin labelling solution for 20 min at 4°C.
4. Quench residual biotin by washing twice with chilled glycine solution, each for 5 min at 4°C.
5. Lyse the cells in 1-ml of extraction buffer at 4°C with gentle agi-tation. Lysis is complete when cell clumps are no longer visible to the eye.
6. Transfer each lysate to a 1.5-ml microcentrifuge tube and centrifuge at 15,000 × g in a cooled (4°C) bench top microcentrifuge 6.3.2. Examining
Steady-State Cell Surface Density by Biotinylation
for 90 min. This clears the lysate of any residual cellular debris (see Note 5).
7. Determine the protein content of each lysate using a cell lysate total protein assay (see Subheading 6.3.2).
8. Dilute the lysates with extraction buffer so that they are all of the same concentration. The lysates should then be stored on ice.
9. Mix equal volumes of each lysate (containing equal quantities of total protein) with 50-µl of neutravidin beads in microcen-trifuge tubes and incubate them with rotation at 4°C for 2 h.
This allows the biotinylated proteins in the lysate to bind to the neutravidin beads.
10. Pellet the neutravidin beads by centrifugation and remove the supernatant (see Note 6).
11. Wash the neutravidin bead pellet four times with extraction buffer to remove any non-specifically bound proteins.
12. Elute the biotinylated proteins from the neutravidin beads by re-suspension of the washed neutravidin bead pellet in 50-µl of reducing sample buffer and incubate for 15 min at room tem-perature.
13. Pellet the neutravidin beads by centrifugation and resolve the eluted proteins contained within the reducing sample buffer by SDS-PAGE (see Subheading 6.3.4).
This protocol describes how the protein content of cell lysates is to be determined using the BCA method. Many alternative methods are available in kit form from numerous suppliers.
1. Pipette 10 µl of each cell lysate into individual wells of a flat-bottomed 96-well plate in duplicate.
2. Pipette 10 µl of each protein standard into individual wells of a flat-bottomed 96-well plate in triplicate.
3. Add 200 µl of BCA reagent to each well and incubate the plate at 37°C for 30 min.
4. The absorbance of each sample at 550 nm is measured using a spectrophotometer.
5. Plot the mean absorbance values of the protein standards against protein concentration to create a calibration curve from which the mean protein content of the cell lysates can be extrapo-lated.
These instructions relate to the use of Bio-Rad protean2 appara-tus but can be easily modified for use with any other SDS-PAGE gel system. The following quantities are sufficient for the produc-tion of two 0.75-mm thick, 10% mini gels. The blotting protocol relates to the use of the Bio-Rad semi-dry transfer cell.
6.3.3. Cell Lysate Total Protein Assay
6.3.4. SDS-PAGE and Western Blotting
1. Clean the front and back gel casting plates with warm water and finally rinse with distilled water. Allow to dry.
2. Place the glass plates into the casting frame.
3. Prepare the separating gel mix: 4 ml of glycerol solution, 3.3-ml acrylamide mix, 2.5-ml resolving buffer, 100-µl SDS solution, 100-µl APS solution and 4-µl TEMED. Gently mix (see Note 7) and pipette into the gap between the two glass plates filling 3/4 of the way up (leaving sufficient room for the stacking gel and spacer comb). Finally overlay the mixture with water-saturated isobutanol. The gel should polymerise within 30 min.
4. Remove the isobutanol layer and rinse the top of the gel with water.
5. Prepare the stacking gel mix: 1.4 ml of glycerol solution, 330-µl of acrylamide mix, 250-µl of resolving buffer, 20-µl of SDS solu-tion, 20-µl of APS solution and 2-µl of TEMED. Gently mix (see Note 7) and pipette into the gap between the two glass plates to the top. Immediately place the spacer comb into place to cast the sample wells. The gel should polymerise within 30 min.
6. Remove the gels from the casting apparatus and assemble the running tank (see Note 8). Fill the central buffer reservoir with the running buffer and carefully remove the combs.
7. Load equal amounts of each protein sample into individual lanes and a single lane with pre-stained protein standards.
8. Half fill the external tank with running buffer and place the lid on the tank.
9. Run the gel at 200 mV for 45 min or until the dye-fronts are run off the bottom of the gel.
10. Disassemble the gel running apparatus and remove one of the glass plates from the gel. Using a sharp blade, remove the stack-ing gel and discard.
11. Remove the gel from the glass plate and submerge it in chilled transfer buffer for no longer than 5 min.
12. Concurrently, soak nitrocellulose membrane cut to the same size as the resolving gel in chilled transfer buffer (see Note 9).
13. Cut two pieces of thick blotting paper to the same size as the resolving gel and soak in chilled transfer buffer.
14. Prepare the Western blot transfer stack as follows: (from the bottom) 1× soaked blotting paper, soaked nitrocellulose membrane, resolving gel and1× soaked blotting paper. This is summarised in Fig. 6.1.
15. Transfer proteins onto nitrocellulose using a Bio-Rad semi-dry transfer cell. Both electrodes must be wetted with transfer buffer. Once on the apparatus, use a sample tube to roll out any air bubbles which may be trapped within the
transfer stack. Assemble the transfer cell and run at 0.03 mA for 90 min.
16. Remove the nitrocellulose membrane from the apparatus and rinse twice with distilled water and twice with PBS-T.
17. Block the membrane by incubating with blocking solution for 1 h at room temperature with gentle agitation.
18. Incubate the membrane with mouse anti-FLAG antibody diluted in antibody dilution buffer overnight at 4°C with gentle agitation.
19. Wash excess antibody off the membrane with PBS-T at room temperature with gentle agitation. Four washes each of 5 min should be sufficient.
20. Incubate the membrane with anti-mouse HRP-conjugated antibodies.
21. Wash excess antibody off the membrane with PBS-T at room temperature with gentle agitation. Four washes each of 5 min should be sufficient.
22. During the final wash step prepare 1 ml of ECL reagent.
23. Place the washed membrane on a clean sheet of acetate and pipette the ECL reagent over the entire surface of the mem-brane. Incubate at room temperature for 2 min.
24. Drain the ECL reagent from the surface and cover the mem-brane with a second sheet of acetate.
25. The acetate containing the membrane is then placed in an X-ray cassette with X-ray film for an appropriate length of time (see Note 10). The film is then submerged in developing fluid until the signal has developed fully and then submerged in fixative fluid to preserve the signal. An example of the data obtained is shown in Fig. 6.2.
Fig. 6.1. Setting up for a Western blot transfer. These instructions assume the use of a Bio-Rad semi-dry transfer cell.
The “Western blot stack” should be assembled as shown. A layer of pre-soaked thick blotting paper is laid flat onto the cathode. The soaked nitrocellulose is then carefully placed on top of this, followed by the soaked SDS-PAGE gel.
The stack is completed by the addition of a final layer of soaked thick blotting paper. Air bubbles are then rolled out of the stack and the anode secured in place. When electrical current is passed through the stack, the SDS-coated proteins will migrate towards the cathode and become immobilised on the nitrocellulose membrane.
1. The Sulpho-S-S-biotin used is impermeable to cell membranes so only extracellularly exposed proteins will be labelled. Label-ling with biotin also requires that lysine residues are accessible to the reagent, so some channel proteins may not be suitable for this type of labelling.
2. Solutions containing non-fat dry milk should be filtered through tissue paper to remove particulate matter. Failure to do this can lead to speckled background signals on the final blot.
3. Quantification of data may be achieved by digitally scanning blots although care must be taken during data acquisition not to saturate the signal. Alternatively automated systems may be used to capture the chemiluminescence signal.
4. The duration of the internalisation steps required will vary between different ion channels and should be determined empirically. For most applications 0, 15, 30 and 60 min should prove to be a good starting point.