Exercise 4.2 - Separation of Protein Standards: SDS-PAGE

LEVEL I

Figure 4.6 Protein standards and SDS-PAGE

Figure 4.7 Rf vs log molecular weight (Laemmli)

Materials

• 10% SDS-polyacrylamide gel from Exercise 4.1
• Protein standards 10
• 2X-SDS Sample Buffer
• 1X-SDS Electrophoresis Running Buffer (Tris-Glycine + SDS)
• 0.001% (w/v) Bromophenol Blue
• Micropipettes with flat tips for electrophoresis wells

Procedure

1. Remove the teflon combs from prepared gels by gently lifting the combs from the chamber. Rinse the wells (formed by the removal of the combs) with distilled water and drain off the water.

2. Fill the wells and the chamber with running buffer.

3. Prepare aliquots of a known protein standard by mixing equal parts of the protein standard with 2X sample buffer.

4. Using a micropipette, add the sample to the bottom of a well. 11

5. Add the same volume used in step 4 of SDS-sample buffer or bromophenol blue to a separate well. 12

6. Remove the gel from its casting stand and assemble it into the appropriate slab unit for running the electrophoresis. Be sure to follow the manufacturer's directions for assembly.

7. Pour a sufficient quantity of running buffer into both the lower and upper chambers of the electrophoresis apparatus until the bottom of the gel is immersed in buffer, and the top is covered, while the electrodes reach into the buffer of the upper chamber. Be careful not to disturb the samples in the wells when adding buffer to the upper chamber.

8. Assemble the top of the electrophoresis apparatus and connect the system to an appropriate power source. Be sure that the cathode (+) is connected to the upper buffer chamber.

9. Turn on the power supply and run the gel at 20 mA constant current per 1.5 mm gel.
   For example, if two gels are run, each having 1.5 mm spacers, the current should be adjusted to 40 mA. One gel with 1.5 mm spacers should be run at 20 mA, while a gel with 0.75 mm spacers should be run at 10 mA.

10. When the tracking dye reaches the separating gel layer, increase the current to 30 mA per 1.5 mm gel.

11. Continue applying the current until the tracking dye reaches the bottom of the separating gel layer (approximately 4 hours).

12. Turn off and disconnect the power supply. Disassemble the gel apparatus and remove the glass sandwich containing the gel. Place the sandwich flat on paper towels and carefully remove the clamps from the sandwich.

13. Working on one side of the sandwich, carefully slide one of the spacers out from between the two glass plates. Using the spacer or a plastic wedge as a lever, gently pry the glass plates apart without damaging the gel contained within.

14. Lift the bottom glass plate with the gel and transfer the gel to an appropriate container 13 filled with buffer, stain or preservative.
    The gel may at this point be used for Coomasie Blue staining (Exercise 4.3 ), silver staining (Exercise 4.4), enzyme detection (Exercise 4.7), Western blots (Exercise 4.9) or for more advanced procedur es, such as electroblotting or electroelution.
    If prestained protein standards were used, the gels may be scanned directly for analysis (Exercise 4.5 or Exercise 4.6). Place the gel into 50% methanol and gently rock the container for about 30 minutes prior to scanning. This can be accomplished by placing the gels into a flat dish and gently lifting the edge of the disk once every 30 seconds. There are commercially available rocker units for this purpose.
    If the gel is to be dried, use a commercial gel dryer, such as the Hoefer SE540 or SE 1160 Slab Gel Dryer. Follow the manufactuer's directions. Figure 4.6 demonstrates a dried and stained gel containing a series of proteins of known molecular weights.

15. Plot the relative mobility of each protein against the log of its molecular weight.

    Relative mobility is the term used for the ratio of the distance the protein has moved from its point of origin (the beginning of the separating gel) relative to the distance the tracking dye has moved (the gel front). The ratio is abbreviated as Rf. Molecular weight is expressed in daltons. Figure 4.7 presents a plot of the relative molecular weight of protein standards against the log of their molecular weight.

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