COOMASSIE BLUE STAINING
Detection of protein bands in a gel by Coomassie blue staining depends on nonspecificbinding of a dye, Coomassie brilliant blue R, to proteins. The detection limit is 0.3 to 1µg/protein band. In this procedure, proteins separated in a polyacrylamide gel areprecipitated using a fixing solution containing methanol/acetic acid. The location of theprecipitated proteins is then detected using Coomassie blue (which turns the entire gelblue). After destaining, the blue protein bands appear against a clear background. The gelcan then be stored in acetic acid or water, photographed, or dried to maintain a permanentrecord.
Materials
Polyacrylamide gel (UNIT 10.2A)
Fixing solution for Coomassie blue and silver staining (see recipe)
Coomassie blue staining solution (see recipe)
Methanol/acetic acid destaining solution (see recipe)
7% (v/v) aqueous acetic acid
Whatman 3MM filter paper (optional)
Gel dryer (optional)
1. Place the polyacrylamide gel in a plastic container and cover with 3 to 5 gel volumes of fixing solution. Agitate slowly 2 hr at room temperature on an orbital shaker orrocking platform.
If agitation is too rapid, the gel may break apart. Use fixing solution only once.
2. Pour out fixing solution. Cover the gel with Coomassie blue staining solution for 4 hr and agitate slowly.Use staining solution only once.
3. Pour out staining solution. Rinse the gel briefly with ∼50 ml fixing solution.
4. Pour out fixing solution. Cover the gel with methanol/acetic acid destaining solutionfor 2 hr and agitate slowly.
5. Pour out destaining solution. Add fresh methanol/acetic acid destaining solution andcontinue destaining until blue bands and a clear background are obtained. Store thegel in 7% aqueous acetic acid or water.
Do not store gels in fixing solution because protein bands will eventually disappear. Gels can be stored up to 1 year at 4°C in a sealable plastic bag.
6. If desired, photograph the gel (see Support Protocol 1).
7. If desired, dry the gel to maintain a permanent gel record. Place the gel on two sheetsof Whatman 3MM filter paper and cover top with plastic wrap. Dry in a conventionalgel dryer 1 to 2 hr at ∼80°C.
Alternatively, place gel on filter paper and dry according to instructions supplied with gelDryer
RAPID COOMASSIE BLUE STAINING
Protein bands stained using this protocol can be detected within 5 to 10 min after addingrapid Coomassie staining solution. Because the Coomassie blue concentration is lowerthan that used in Basic Protocol 1, the gel background never stains very darkly and thebands can be seen even while the gel remains in the staining solution. Another differenceis that isopropanol is substituted for methanol in the fixing solution. This method isslightly less sensitive than the standard procedure (see Basic Protocol 1).
Additional Materials (also see Basic Protocol 1)
Isopropanol fixing solution (see recipe)
Rapid Coomassie blue staining solution (see recipe)
10% (v/v) acetic acid
1. Place a polyacrylamide gel in a plastic or glass container. Cover the gel with 3 to 5gel volumes isopropanol fixing solution and shake gently at room temperature. Fora 0.7-mm-thick gel, shake 10 to 15 min; for a 1.5-mm-thick gel, shake 30 to 60 min.
2. Pour out fixing solution. Cover the gel with rapid Coomassie blue staining solutionand shake gently until desired intensity is reached, 2 hr to overnight at roomtemperature.
Bands will become visible even in the staining solution within 5 to 30 min, depending on gel thickness. The gel background will never stain very darkly.
3. Pour out staining solution. Cover the gel with 10% acetic acid to destain, shakinggently ≥2 hr at room temperature until a clear background is obtained.
4. If necessary, pour out 10% acetic acid and add more. Continue destaining until clearbackground is obtained. Store gel in 7% acetic acid or water, or in plastic wrap at4°C.
It is usually unnecessary to add additional destaining solution.
5. If desired, photograph (see Support Protocol 1) or dry (see Basic Protocol 1, step 7)the gel.
REAGENTS AND SOLUTIONS
Use high-quality deionized, distilled water (≥18 MΩ) in all recipes and protocol steps. For common stocksolutions, see APPENDIX 2
Coomassie blue staining solution
50% (v/v) methanol
0.05% (w/v) Coomassie brilliant blue R-250 (Bio-Rad or Pierce)
10% (v/v) acetic acid
40% H2O
Dissolve Coomassie brilliant blue R in methanol before adding acetic acid and water.Store for up to 6 months at room temperature. If precipitate is observed followingprolonged storage, filter to obtain a homogeneous solution.
Drying solution
10% (v/v) ethanol
4% (v/v) glycerol
86% H2O
Store up to ∼1 month at room temperature
Fixing solution for Coomassie blue and silver staining
50% (v/v) methanol
10% (v/v) acetic acid
40% H2O
Store up to ∼1 month at room temperature
Isopropanol fixing solution
25% (v/v) isopropanol
10% (v/v) acetic acid
65% H2O
Store indefinitely at room temperature
Methanol/acetic acid destaining solution
5% (v/v) methanol
7% (v/v) acetic acid
88% H2O
Store up to ∼1 month at room temperature
Rapid Coomassie blue staining solution
10% (v/v) acetic acid
0.006% (w/v) Coomassie brilliant blue G-250 (Bio-Rad)
90% H2O
Store indefinitely at room temperature
COMMENTARY
Background Information
Coomassie brilliant blue (Basic Protocol 1)binds nonspecifically to proteins (Wilson,1983). Because the dye does not bind to thepolyacrylamide gel, proteins will be detectedas blue bands surrounded by clear gel zones.
Silver staining relies on differential reduction of silver ions, which is the basis for photographic processes. A highly sensitive photochemical silver staining technique (Switzer etal., 1979; Merril et al., 1984) permits the detection of polypeptides in gels at more than 100×lower concentrations than Coomassie brilliantblue (i.e., femtomole levels of protein).
Fluorescent protein gel stains provide anumber of advantages over conventional colorimetric stains. The SYPRO Orange and Redprotein gel stains described here can detect 1 to2 ng protein per minigel band, more sensitivethan Coomassie brilliant blue staining and assensitive as many silver staining techniques. Inaddition, staining is complete in <1 hr. Afterelectrophoresis, the gel is simply stained,rinsed, and photographed; no separate fixationor destaining step is required and there is nofear of overstaining the gel (Steinberg et al.,1996a,b, 1997). In addition, stained proteinscan be visualized using a standard 300-nm UVtransilluminator or a laser scanner (Fig. 10.6.2).
Because the dyes interact with the SDS coataround proteins in the gel, they give more consistent staining between different types of proteins compared to Coomassie or silver stainingand do not exhibit negative staining. Furthermore, the dyes detect a variety of proteins downto ∼6500 Da without staining nucleic acid orlipopolysaccharide contaminants that aresometimes found in protein preparations derived from cell or tissue extracts.
Anticipated Results
The sensitivity of Coomassie blue gel staining is 0.3 to 1 µg/protein band; the sensitivityof silver staining is 2 to 5 ng/protein band. Thesensitivity of both stains varies in an unpredictable manner with the protein being stained.
For fluorescent dyes, detection limits aretypically ∼500 ng protein/band in room light,∼50 ng protein/band with 300-nm transillumination, and ∼1 to 2 ng protein/band in a photograph taken with Polaroid 667 black-and-whiteprint film. The authors achieve detection limitsof 1 to 2 ng/band using a Fotodyne Foto/UV450 ultraviolet transilluminator, which has six15-watt bulbs that provide peak illumination at312 nm. When using weaker illuminationsources, exposures must be correspondinglylonger. Although the authors’ detection limitsare 1 to 2 ng/band for most proteins, it shouldbe emphasized that bands containing 5 to 10ng/protein are more readily detected. Bandscontaining less than 5 to 10 ng protein requirelonger exposures and sharp bands for goodvisualization. Longer exposures can result inhigher background.
Time Considerations
Coomassie blue staining requires 8 to 12 hr. Fixation may beextended for several days before Coomassieblue staining.
Use of either of the rapid staining protocolsconsiderably reduces the time required to visualize proteins. Detection of protein bands byrapid Coomassie blue staining requires ≤90 minfrom the time a minigel is run (30 to 60 min)until the gel is fixed (10 min) and placed instaining solution (5 to 10 min); however, additional time may be necessary for larger gels.