The Biotechnology Project: Staining the Gel

Staining the Gel

Polyacrylamide Gel Electrophoresis image

Figure 5.3 Polyacrylamide Gel Electrophoresis (PAGE) a) The gel is poured vertically between two glass plates. b.) Protein bands are separated on the basis of relative molecular weight and visualized with stains. Figure from Seidman and Moore. Basic Lab Methods for Biotechnology. Prentice Hall, New Jersey

There are several methods for visualizing the proteins in polyacrylamide gels. These staining methods are not specific for the target protein- they stain all proteins. Coomassie Blue Stain is the most common method and it works well when the detection of minor bands is not necessary. Generally, 10-20 µg protein/cm2/band provides good detection by Coomassie staining. When sensitivity is important, silver staining is the method of choice because it can detect bands that contain nanogram quantities of protein. Proteins in acrylamide gels can also stained with heavy metal salts. While not as sensitive as silver staining, heavy metal staining has the advantage that it allows quantitative recovery of resolved proteins from the gel.

Method A. Coomassie Blue Staining

Transfer your gel to a Pyrex dish containing Coomassie Blue Stain. The recipe for the stain is found in Appendix A. Stain your gel for approximately 1 hour; overnight staining will also work but it will take longer to destain. Do not use plastic dishes because they permanently take up the stain.
Transfer your gel to destain solution 1 (50% methanol and 10% acetic acid) and incubate for approximately 30 minutes. Destaining can be left overnight or longer. To speed up destaining add a small piece of foam (or even a wadded up kimwipe) to the destaining tray. It will absorb the stain.
Note: If the gel shrinks too much to view the bands, you may swell it by putting the gel in DI water with 1% glycerol for 30 minutes or so.
View the gel and interpret your results. Make a diagram in your notebook or take a picture. Alternatively, you may dry the gel according to the procedure below.

Drying the gel:

Kits for air drying gels are commercially available at a nominal cost and these work quite well, although overnight drying is usually required. Follow the directions on the kit for setting up the apparatus.

For all gel drying methods, soak the gel for 30 minutes in water containing 1% glycerol to prevent cracking of the gel during the drying process.

Method B. Silver Staining:

Read the Bio-Rad silver stain directions and watch the instructional video.
You will need to prepare five different solutions for this procedure: fixatives, oxidizer, silver stain reagent, developer and stop solution. You may make the fixatives, developer and stop solution ahead of time but you must prepare the oxidizer and silver stain the day that staining is performed. Prepare the reagents as follows.

Fixatives and Stop Solution. The recipes for the fixatives and stop solution are contained in Table 3 (not in the appendix).

Oxidizer and Silver stain. The concentrates for the oxidizer and silver stain are provided in the kit and must be diluted 1/10 in water. Suggested volumes are provided in the kit.

Developer. The concentrate for the developer is also provided in the kit and must be diluted in water. To prepare the developer dissolve one bottle of concentrate in 3.6 liters of deionized water by stirring for 15 minutes at room temperature. This is enough for 12 mini gels like those we are using. To prepare smaller amounts, use 32 grams of developer per liter of deionized water. Be sure to shake the bottle thoroughly before weighing out the concentrate since the components settle out. Failure to mix the contents can result in very slow or no development. Store the diluted developer solution at room temperature for up to 1 month. Keep the solution tightly covered to avoid evaporation of paraformaldehyde. Use the volumes for mini gels.
Perform silver staining as shown in Table 5.3 below. Do not start the procedure until you have the stop solution ready. We have observed that students have a tendency to overstain. Gels that are overstained will have high background or surface deposits of silver. We advise you to monitor development of your gel closely.

Table 5.2 Reagents and Times for Silver Staining.
Reagents	Volumes	Times
1. Fixative (40% methanol/10% acetic acid)	200 ml	30 min
2. Fixative (10% ethanol/5% acetic acid)	200 ml	15 min
3. Fixative (10% ethanol/5% acetic acid)	200 ml	15 min
4. Oxidizer	100 ml	5 min
5. Deionized water (milli-Q)	200 ml	5 min
6. Deionized water (milli-Q)	200 ml	5 min
7. Deionized water (milli-Q)	200 ml	*
8. Silver Reagent	100 ml	20 min
9. Deionized water (milli-Q)	200 ml	1 min
10. Developer	100 ml	**
11. Developer	100 ml	5 min
12. Developer	100 ml	5 min
13. STOP (5% acetic acid)	200 ml	5 min
* Repeat washes 5, 6 and 7 until all the yellow color has been removed from the gel. ** Develop 30 seconds or until the solution turns yellow or until a brown "smoky" precipitate appears. Then pour off the developer, proceed to the next step and add fresh developer.

View the gel and interpret your results. Sketch, photograph or dry the gel.

Chapter5: Procedure 5.3

Staining the Gel

Method A. Coomassie Blue Staining

Drying the gel:

Method B. Silver Staining:

Chapter5:
Procedure 5.3