Materials

1. 30% Acrylamide: 29.2% acrylamide/0.8% to-acrylamide in H2O (see Note 1).

2. H2O-saturated isobutyl alcohol (see Note 2).

6. 25% ammonium persulfate (APS; see Note 5).

8. 2X SDS sample buffer: 100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 5% 2-mercaptoethanol (2-ME), 2 mM EDTA, 0.1 mg/mL bromophenol blue (see Note 6).

9. 5X Electrophoresis buffer: 15.1 g Tris base, 72 g glycine, and 5 g SDS in 1 L of H2O.

10. Prestained molecular-weight (MW)-marker mixture (e.g., Kaleidoscope standards from Bio-Rad).

11. Electrophoresis system: Bio-Rad Mini-PROTEAN 3 or equivalent; 0.75-mm spacers are recommended.

12. Power supply capable of providing constant voltage of 150 V or higher.

13. 5X transfer buffer: 15.1 g Tris and 72 g glycine in 1 L H2O.

14. 1X transfer buffer: Mix 300 mL of H2O, 100 mL of 5X transfer buffer, 100 mL of methanol, and 1.9 mL of 10% SDS.

15. Semidry transfer apparatus (e.g., Trans-Blot SD, Bio-Rad).

16. Nitrocellulose membrane (e.g., Protran nitrocellulose membrane BA-85, S&S).

17. Extra-thick blot paper (e.g., Bio-Rad); 2-3 mm thickness.

18. Ponceau S staining solution: 0.25% (w/v) Ponceau S in 1% acetic acid.

19. Primary antibody: antigen-specific.

20. Secondary antibody-horseradish peroxidase conjugate (e.g., Jackson Immuno-laboratories): species-specific for the animal in which the primary antibody has been raised.

22. Tris-buffered saline-Tween-20(TBS-T): 0.9% NaCl, 20 mM Tris-HCl, pH 7.5, and 0.05% Tween-20.

23. Blocking and antibody dilution solution: dissolve 5 g of nonfat dry milk in 100 mL of TBS-T.

24. 10% Thimerosal (a preservative).

25. Chemiluminescence reagent (e.g., ECL, Amersham).

27. Stripping solution: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM 2-ME.

3. Methods 3.1. SDS-PAGE

1. Prepare samples as described in the protein extraction method of your choice. Mix the extracts with 1 vol of 2X sample buffer and boil at 95°C for 3 min (see Note 7). Centrifuge samples at 12,000g for 30 s after boiling to pellet undis-solved particles and bring down moisture from the wall of the tube into the solution. Allow the samples to cool down to room temperature before loading onto a gel.

2. Assemble a glass plate sandwich on a casting stand according to instructions provided by the manufacturer of the electrophoresis system. The two glass plates will be separated by spacer strips at two opposite edges, and the gel will be poured into the space between the plates.

3. Prepare resolving and stacking gel solutions according to Table 1. Volumes given are for two gels with the Bio-Rad Mini-Protean system. Volumes should be adjusted, if different systems are used.

4. Add 15 ^L of 25% APS and 10 ^L TEMED to the resolving solution and use immediately. Acrylamide should begin to polymerize within 5 min.

5. Pour the gel solution into the sandwich with a pipet until the height of the solution reaches three-fourths of that of the small glass plate (see Note 8).

6. Overlay the solution with 500 ^L of H2O-saturated isobutanol. Be careful not to disturb the surface of the solution (see Note 9).

7. Let the solution polymerize for 30 min. A line will be visible between the polymerized gel and the isobutanol layer because of differential light transmission of the two layers (see Note 10).

8. Pour off any liquid from the top of the gel and rinse the surface with H2O. Remove H2O as much as possible. Remaining H2O may cause the stacking gel to shrink.

Table 1

Recipes for Resolving and Stacking Gel Solutions

% Polyacrylamide (resolving gel)

6 B 10 15 Stacking gel

30% Acrylamide/

bis-acrylamide (mL) 1 M Trus-HCl, pH B.B (mL) l M Tris-HCl, pH 6.B (mL) lO% SDS (mL) H2O (mL) Total volume (mL)

This recipe produces enough gel solutions to make two mini-gels for Bio-Rad Mini-PROTEAN 3 system. If a different system is used, adjust the volumes of the solutions according to instruction manuals.

All solutions must be prepared with Milli-Q-purified or double-distilled H2O, and filtered through a 0.45-^m filter.

After all components are added, mix the solution gently. Be careful not to make foam.

9. Add 7.5 ^L of 25% APS and 7.5 ^L TEMED to the stacking gel solution and pour immediately into the glass plate sandwich, until the solution reaches the top of the small glass plate. Pay attention not to introduce bubbles. The solution should start to polymerize within 5 min.

10. Insert a 0.75-mm comb into the sandwich. If bubbles are trapped below the comb, remove the comb, add more stacking gel solution to the top of the small glass plate, remove bubbles, and insert the comb again. Because acrylamide polymerizes quickly, the whole procedure should be done as quickly as possible.

11. Allow the stacking gel to polymerize for 30 min. Make sure that the gel has polymerized by checking the leftover solution in the original container (see Note 10).

12. Attach the glass sandwich to the electrophoresis system according to the instruction manual.

13. Pour 1X electrophoresis buffer into the upper buffer chamber and the lower chamber. The top and bottom of the gel should be submerged in buffer for electric current to flow through the gel matrix. If excessive bubbles are trapped in the bottom of the gel, they should be removed by a syringe with a curved needle (see Note 11).

14. Remove the comb carefully, making sure not to disrupt wells. Rinse the wells with 1X electrophoresis buffer using a syringe with a thin needle to get rid of gel pieces that may be present. These gel pieces can result in uneven migration of proteins.

15. Load samples and a mixture of prestained MW markers into separate wells (see Notes 12 and 13). The mixture of MW markers should be also boiled until the precipitate dissolves completely. If possible, use an equal volume of samples across the lanes on a gel (see Note 14). Load an equal volume of 1X sample buffer into unused wells.

16. Check buffer levels before connecting the electrophoresis cell to a power supply. The bottom and top of the gel, and both cathode and anode electrodes, should be submerged under the electrophoresis buffer.

17. Make sure that the power supply is off before it is connected to the electrophoresis tank. Connect the electrode of the upper buffer chamber to the cathode (-) outlet of the power supply and that of the lower buffer chamber to the anode (+) outlet.

18. Set the power supply at 150 V of constant voltage and start running (see Note 15).

19. Stop the power supply when bromophenol blue dye reaches the bottom of the gel or a desired resolution is achieved (see Note 16).

20. Remove the glass sandwich from the electrophoresis tank and take off one of the two glass plates. Do not take off the gel from the other glass plate because it is easier to handle the gel while it is attached. Be careful not to tear the gel.

21. Remove the stacking gel along with the top 1 to 2 mm of the separating gel. It is difficult to align a whole gel on a blot membrane because the stacking gel is sticky and will adhere to the membrane.

3.2. Electroblotting

1. Place the gel along with the glass plate in a tray containing 1X transfer buffer.

2. Remove the gel gently from the glass plate. The gel usually comes off the glass plate when the glass plate is gently shaken in the transfer buffer.

3. Prepare a nitrocellulose membrane and two layers of blot paper. Each blot paper layer should be 2- to 3-mm thick (see Note 17). Cut the membrane and the blot paper so that their length and width are each 1 cm larger than the gel. If two gels are transferred together on the same membrane, double the area of the membrane and the blot paper. Wet the membrane and blot papers with 1X transfer buffer.

4. Sandwich the gel and the membrane between the two blot paper layers and arrange this sandwich on the anode plate of the semidry blotting apparatus as shown in Fig. 1 (see Note 18).

5. Remove bubbles from the gel-membrane blot paper sandwich by rolling a plastic pipet on the top blot paper from one end to the other. Repeat this in the other direction. Do not push too hard while rolling the pipet; it may squeeze the gel out of the sandwich.

6. Wipe off transfer buffer from the surrounding area of the sandwich.

7. Place the cathode plate on the sandwich. Avoid sideways movement of the cathode plate, as it may misalign the sandwich.

8. Connect the blotting apparatus to a power supply. Do not switch the polarity. Unlike SDS-PAGE, electroblotting requires low voltage and high current. A power supply for electroblotting should be able to produce 2 A or higher.

9. Set the power supply at 20 to 23 V of constant voltage and start running (see Note 19).

10. Stop the power supply and take off the upper electrode carefully.

11. Remove the top blot paper and the gel. Check remaining prestained markers on the gel and transferred markers on the membrane. If most of the markers were transferred, it indicates that most of your proteins of interest were also transferred.

12. Wash the membrane briefly with TBS-T.

13. Remove TBS-T and add Ponceau S solution just enough to cover the membrane.

14. Shake the tray by hand for a couple of minutes. The protein bands should be readily visible. Visually assess the efficiency of the transfer (see Note 20).

15. Remove the Ponceau S solution and wash the membrane with TBS-T until the staining is completely washed off.

16. Add blocking solution and incubate at room temperature for 30 min.

3.3. Immunodetection

1. Remove the blocking solution and add the primary antibody diluted in blocking solution (see Notes 21 and 22).

2. Incubate the primary antibody at room temp for 2 to 3 h or at 4°C overnight with gentle shaking.

3. Remove the primary antibody (see Note 23).

4. Wash the membrane with TBS-T at room temp for 10 min. Repeat this procedure three times.

5. Add secondary antibody diluted in blocking solution. Incubate at room temperature for 1 h with gentle shaking.

6. Remove the secondary antibody.

7. Wash the membrane with TBS-T at room temperature for 10 min. Repeat this procedure six to eight times.

8. After the final wash, drain TBS-T as much as possible and add a chemilumines-cent reagent (e.g., ECL, Amersham).

9. After 1 to 2 min incubation, pick up the membrane with a pair of forceps and drain the chemiluminescent reagent as much as possible by allowing the membrane to touch an absorbent such as a paper towel.

10. Put the membrane between two sheets of plastic wrap to prevent films from getting wet.

11. Record the signal by exposing the blot to an X-ray film in a dark room or by using a charge-coupled device imaging system. Signals should be visible within 30 min (see Note 24).

12. If necessary, the blot can be stripped and reprobed with a primary antibody against a different antigen, saving time and samples (see Note 25).

4. Notes

1. Dissolve 29.2 g acrylamide and 0.8 g bs-acrylamide in 70 mL H2O, add H2O to 100 mL, and filter the solution with a 0.45-pm filter. Acrylamide is light-sensitive. The container should be covered with foil or otherwise shielded from light. Acrylamide is also a neurotoxin. When weighing acrylamide powder, a mask and gloves should be worn. When handling acrylamide solution, gloves should be worn.

2. Mix 1 vol of isobutyl alcohol and 1 vol of H2O by a vigorous shaking and allow to stand overnight. The top layer is water-saturated isobutyl alcohol and the bottom layer is water. Use only the top layer.

3. Filter the solution with a 0.45-pm filter.

4. pH may change during storage. If pH changes more than 0.5, discard and make a fresh batch.

5. Dissolve 2.5 g APS in 8 mL H2O, add H2O to 10 mL, filter (0.45 pm) and make 1-mL aliquots. Store a working aliquot at 4°C and the rest of the aliquots at -80°C.

7. SDS and 2-ME will denature proteins and reduce intra- or intermolecular disul-fide bonds, which also inactivates most, if not all, proteases present in the extracts. After boiling, the samples can be stored at -80°C indefinitely and repeatedly thawed and frozen. If protein samples were already mixed with 2X sample buffer and are taken from -80°C, they just need to be boiled for 1 min or until precipitate dissolves completely.

8. Care should be taken in avoiding to introduce bubbles, as the solution contains SDS, which is a detergent and prone to foaming.

9. The H2O-saturated isobutanol layer ensures that the top of the gel is flat after polymerization.

10. If the gel does not polymerize within 30 min, 25% APS and/or TEMED should be replaced with fresh aliquots or fresh reagents should be purchased.

11. If there is a leak from the upper buffer chamber, it should be fixed or the system will have to be reassembled. Insufficient buffer in the upper chamber can cause partial overheating of the glass plates, which can lead to breakage of the glass plates during the run. It is always safer to monitor the level of the upper buffer during run and replenish the buffer if necessary.

12. When 10 well combs are used in the Bio-Rad Mini-PROTEAN system, 20 to 50 ^g total protein is recommended. If too much protein is loaded, resolution will be poor.

13. Samples can be loaded using either a Hamilton syringe or a pipettor with a disposable gel-loading tip. The tip of the needle or the disposable tip should be thin enough to be inserted between the two glass plates. When samples are applied, the tip of a needle or a pipet tip should be as close as possible to the bottom of the well. This minimizes mixing of the sample with electrophoresis buffer during loading.

14. If too much or too little sample volume is used compared with adjacent wells, protein samples will spread into adjacent wells or will be compressed by protein samples in adjacent wells, respectively. If the volume of a sample is less than half of that in adjacent lanes, add 1X SDS sample buffer to normalize the volume.

15. It is more convenient to use constant voltage than constant current because the voltage does not need to be changed according to the number of gels run using the same power supply. Current will be proportionally increased as the number of gels connected to the power supply increases. If current reads too high or too low, it is most likely that there is a bad connection or that the electrophoresis buffer was not correctly made.

16. An adequate percentage of polyacrylamide should be used to obtain well-resolved Western blot results. This is particularly important to detect different isoforms of clock proteins as a result of phosphorylation. The following is recommended:

6%: proteins of MW 100 kDa or more.

17. If the blot paper is too thin, the transfer buffer will dry out during the procedure. If paper that is thinner than 2 to 3 mm is used, stack multiple sheets together to achieve the appropriate thickness.

18. If prestained markers were run on the gel, there is no need for marking the membrane for lane orientation. If two gels are transferred on the same membrane, each gel can be identified by using different amounts of prestained markers or using different lanes for the markers on two gels.

19. It is more convenient to use constant voltage than constant current because the current will need to be adjusted according to the number of gels being electroblotted. When the appropriate percentage of polyacrylamide and the Bio-

Rad apparatus are used, all the known mammalian clock proteins can be successfully transferred at 23 V within 30 min.

20. If uneven transfer is observed, check the anode and cathode plates. They can distort or sag over a long period of use. This can cause uneven transfer of proteins. If this occurs, replace the defective plate(s) with new one(s).

21. To save primary antibody, the membrane can be trimmed or cut into pieces. Moreover, heat-sealable plastic bags can be used instead of trays.

22. If the size of two clock proteins is substantially different, they can be assayed at the same time using a single gel. Run SDS-PAGE long enough to separate the two proteins by a reasonable distance. Transfer proteins to a membrane, stain the membrane with Ponceau S and cut the membrane between the expected positions of the two clock proteins using the prestained MW markers as a reference.

23. If primary antibody is to be used more than once, add thimerosal to the solution. Make a 10% thimerosal stock solution and add it to the primary antibody solution to make 0.1% thimerosal. Freeze primary antibodies in dry ice and store them at -80°C.

24. If no signal (including background signal) is visible, it is most likely that either the primary or the secondary antibody have not been added, or that the secondary antibody is not compatible with the primary antibody. This could happen, for example, if the primary antibody was generated in rabbits, and the secondary antibody was generated against rat IgG.

25. Wash the blot with TBS-T twice before incubating it in stripping solution. Incubate the blot in stripping solution at 50°C for 30 min with gentle shaking. Remove the stripping solution. Add TBS-T and incubate at room temperature for 10 min. Repeat this step three times to remove remaining SDS and 2-ME from the blot. Incubate the blot in blocking solution at room temperature for 30 min. The blot is ready for incubation with a different primary antibody. After the second immunodetection, the blot can be used again for a third time. However, signal intensity will be significantly reduced after each stripping compared with a fresh blot.

References

1. Bae, K., Lee, C., Hardin, P. E., and Edery, I. (2000) dCLOCK is present in limiting amount and likely mediates daily interactions between the dCLOCK-CYC transcription factor and the PER-TIM complex. J. Neurosci. 20, 1746-1753.

2. Denault, D. L., Loros, J. J. and Dunlap, J. C. (2001) WC-2 mediates WC-1-FRQ interaction within the PAS protein-linked circadian feedback loop of Neurospora. EMBO J. 20, 109-117.

3. Lee, C., Etchegaray, J. P., Cagampang, F. R. A., Loudon, A. S. I., and Reppert, R. M. (2001) Posttranslational mechanisms regulate the mammalian circadian clock. Cell 107, 855-867.

4. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

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