1.4. Western blotting and immunodetection of proteins separated by SDS-PAGE
Western blotting, also known as immunoblotting, refers to the transfer of proteins from a polyacrylamide gel onto a solid support, such as a nitrocellulose, polyvinylidene difluoride (PVDF), or cationic nylon membrane. This membrane is then used in an immunodetection procedure to reveal specific protein(s).
The transfer of proteins from polyacrylamide gels to membranes was originally described by Towbin et al. (1979). The original method uses a tank containing a large volume of transfer buffer and is referred to as tank blotting. Subsequently, special western blotting systems were developed for semi-dry transfer. These systems are equal in performance, thus preference will depend on already available laboratory equipment.
After transfer, unspecific binding sites of the membrane are first blocked by excess protein added to the incubation buffer to suppress nonspecific adsorption of antibodies. Subsequently, the immobilized proteins are reacted with a specific polyclonal or monoclonal antibody. Antigen-antibody complexes are finally revealed through a secondary antibody and chromogenic or chemiluminescent reactions.
The following protocol uses a tank blotting system and a horseradish peroxidase-conjugated secondary antibody of an enhanced chemoluminescence (ECL) detection system (GE Healthcare) for revealing antigen-antibody complexes:
1. Prepare the western blotting transfer buffer:
1.1. 25 mM Tris,
1.2. 192 mM glycine,
1.3. 20 % (v/v) methanol.
2. Cut the PVDF membrane and filter paper sheets to fit the size of the separating gel.
Be sure to handle the PVDF membrane using powder free gloves and forceps.
3. Activate the membrane in methanol for 1–2 min.
4. Immerse in water for 1 min to remove the activating solvent.
5. Incubate the PVDF membrane in transfer buffer for 2 min.
6. Assemble the blotting sandwich (Fig. 3) in the following order in a tray containing western transfer buffer (make sure to keep all items submersed and avoid including air bubbles, especially so between the gel and the membrane):
6.1. A stiff plastic supporting grid.
6.2. A foam sponge or Scotch-Brite pad (3M).
6.3. Two sheets of thick filter paper.
6.4. The polyacrylamide gel.
6.5. The PVDF membrane.
6.6. Two sheets of thick filter paper.
6.7. A foam sponge or Scotch-Brite pad (3M).
6.8. The second stiff plastic supporting grid.
7. Fill the transfer tank with western transfer buffer.
8. Insert the sandwich into the support holder of the blotting apparatus.
Make sure the orientation is correct, as transfer is from the cathode (-) to the anode (+), thus, the gel should face towards the cathode and the membrane face the anode.
9. Connect tank to a high voltage power supply.
This is not your usual electrophoresis power supply, but one that can go up to 200 V, 2000 mA and 200W).
10. Run transfer at a setting of 30 V for about 2.5 h at room temperature.
11. Shut down power supply, disconnect cables.
12. Dismount the blotting sandwich.
13. Mark with a pencil the side of the membrane that faced the gel.
To verify transfer efficiency, the gel can be stained with Coomassie Brilliant Blue after blotting (see section 1.3.3); alternatively, the membrane can be stained with Ponceau S solution [0.5 g of Ponceau S in 100 mL of 1% (v/v) acetic acid aqueous solution] for 2 min, washed two to three times in water and then further destained in water. The PVDF membrane can either be used directly for immunodetection, as described below, or air-dried for later detection (this will require reactivation by immersion in methanol, as described in step 3 of the above list).
1. Prepare 1l of a 10 x PBS stock solution:
1.1. 80 g NaCl,
1.2. 2 g KCl,
1.3. 14.4 g Na2HPO4,
1.4. 2.4 g KH2PO4,
1.5. in 1l ddH2O.
2. Prepare a blocking solution containing 250 ml of buffer A:
2.1. 3.027 g Tris,
2.2. 0.147 g CaCl2,
2.3. 2.33 g NaCl,
2.4. Adjust pH to 8.5,
2.5. 50 g non-fat dried milk,
2.6. Complete the volume to 500 ml with ddH2O.
3. Block unspecific binding sites by immersing the membrane in this solution for 1 h at room temperature on an orbital shaker. Alternatively, membranes may be left in the blocking solution overnight in a refrigerator.
4. Briefly rinse the membrane with two changes of wash buffer (500 µl Tween 20 in 1l of 1 x PBS (make up from the 10 x stock, described in step 1, and adjust the pH to 7.2).
5. Appropriately dilute the primary antibody in blocking solution.
The dilution factor must be determined empirically for each antibody, e.g. through dot blotting of a serial dilution of the antibody.
6. Incubate the membrane in diluted primary antibody for 1 h at room temperature on an orbital shaker.
7. Briefly rinse the membrane with two changes of wash buffer.
8. Keep the membrane in wash buffer for 15 minutes at room temperature.
Use >4 ml of wash buffer per cm2 of membrane.
9. Wash the membrane a further three times for 5 min each, in changes of wash buffer.
10. Dilute the horseradish peroxidase (HRP)-conjugated secondary antibody of the ECL kit in wash buffer.
Again, the dilution factor must be determined empirically for each antibody - a 1:12,000 (v/v) dilution may usually be appropriate.
11. Incubate the membrane in the diluted secondary antibody for 1 h at room temperature on an orbital shaker.
12. Briefly rinse the membrane with two changes of wash buffer.
13. Wash the membrane in >4 ml/cm2 of wash buffer for 15 min at room temperature.
14. Wash the membrane a further three times for 5 min each, in changes of wash buffer.
15. Proceed with the detection reaction to obtain the chemoluminescent signal following the manufacturer’s instructions.
This procedure is specific for each commercial ECL kit.
16. Wrap the blots wetted with ECL solution in household PVC foil and place, protein side up, in an X-ray film cassette.
17. In a dark room place a sheet of autoradiography film on top of the membrane previously wrapped in foil.
18. Close the cassette and expose for a short time, usually 5 min.
19. Immediately develop this first film using commercial X-ray film developer or, if available, an automatic developer system.
20. Based on the obtained band intensity, estimate an optimal exposure
time for a second (or third) film.