In electrophoresis, proteins are separated in an electric field by virtue of their charge and size. Electrophoresis of macromolecules is normally carried out by applying a thin layer of a sample to a solution stabilized by a porous matrix. The matrix can be composed of a number of different materials including paper, cellulose, acetate, or gels made of starch, agarose, or polyacrylamide. Agarose and polyacrylamide can act as a size-selective sieve in the separation. However, polyacrylamide is the most common matrix for separating proteins, probably due to its versatile applications.
In electrophoresis, proteins are separated in an electric field by virtue of their charge and size. Electrophoresis of macromolecules is normally carried out by applying a thin layer of a sample to a solution stabilized by a porous matrix. The matrix can be composed of a number of different materials including paper, cellulose, acetate, or gels made of starch, agarose, or polyacrylamide. Agarose and polyacrylamide can act as a size-selective sieve in the separation. However, polyacrylamide is the most common matrix for separating proteins, probably due to its versatile applications.
The movement of molecules in electrophoresis is dependent on the applied voltage (V), which equals the product of current (I) and resistance (R).
The following power equations are also used in electrophoresis:
where P = power, which provides amount of heat produced in the circuit.
In electrophoresis, voltage and current are supplied by a DC (direct current) power supply, and the electrodes, buffer, and gel are considered to be resistors. Power supply is used to hold one electrical parameter (current, voltage, or power) constant. Most power supplies have more than one pair of outlets. When two gels are connected in parallel to one outlet of a power supply, gel currents are additive. When two gels are connected in series to one outlet of a power supply, gel voltages are additive. Gel currents are additive when two gels are connected in parallel to adjacent outlets of a power supply.
Choice of Driving Force: Constant Current or Constant Voltage?
The resistance of the circuit does not remain constant during electrophoresis. For example, in a discontinuous system (separating and stacking gels) of SDS-PAGE running at constant current, resistance increases. Therefore, voltage will increase over time, leading to increased heat generation and may require active heat removal. When running SDS-PAGE at constant voltage, current drops as the resistance increases. This will not result in a high increase of heat, since the main determinant factor (square root of current) is decreased, although resistance is increased. In contrast, in a continuous system (only separating gel) of SDS-PAGE, resistance decreases, resulting in a heat gain when running at constant voltage.
Figure 3.1 Polymerization of acrylamide.
Polyacrylamide (Figure 3.1) gels are formed by copolymerization of acrylamide monomer, CH2 = CH-CONH2, and a cross-linking comonomer, N,N′-methylenebi-sacrylamide, CH2 = CH-CO-NH-CH2-NH-CO-CH = CH2 (bisacrylamide).
Mechanism of Gel Formation
The mechanism of gel formation is vinyl addition polymerization and is catalyzed by a free radical-generating system composed of ammonium persulfate (the initiator) and an accelerator, N,N,N′,N′-tetramethylethylenediamine (TEMED). TEMED catalyzes the decomposition of the ammonium persulfate to yield a free radical (unpaired electron), which activates the acrylamide monomer. The activated monomer then reacts with an unactivated monomer to begin the polymer chain elongation as shown below:
If S represents SO4 -., its reaction with acrylamide monomer (A) can be written as follows:
and so on.
During the polymer chain elongation, bisacrylamide is randomly cross-linked, resulting in closed loops and a complex “web” polymer (see Figure 3.1) with a characteristic porosity, which depends on the polymerization conditions and monomer concentrations.
In some applications (e.g., acid urea PAGE), riboflavin (or riboflavin-5′-phosphate) is used as an initiator of polymerization of acrylamide, as ammonium persulfate interferes with the stacking of the protein. In the presence of light and oxygen, riboflavin is converted to its leuco form, which is active in initiating polymerization.
Oxygen, a radical scavenger, interferes with polymerization, so that proper degassing to remove dissolved oxygen from acrylamide solutions is crucial for reproducible gel formation.
The effective pore size depends on the acrylamide concentration of a gel. The pore size decreases as the acrylamide concentration increases. Usually, gels are characterized by the two parameters, %T and %C, where %T refers to the total monomer (acrylamide + cross-linker), and %C is the ratio of cross-linker (i.e., bisacrylamide) to acrylamide monomer (w/w). The following formulas are used to calculate:
The effective pore size is established by the three-dimensional network of fibers and pores that are formed by cross-linking acrylamide with bifunctional bisacrylamide.
When the gel is poured into a tube or slab mold, the top of the solution forms a meniscus. If the meniscus is ignored, the gel will polymerize with a curved top, which will cause the separated sample bands to have a similar curved pattern. To eliminate the meniscus, a thin layer of water, water-saturated n-butanol, or isopropanol is carefully floated on the surface of the gel mixture before it polymerizes. The layer of water or water-saturated butanol should be deaerated; otherwise, it will inhibit polymerization on the gel surface.
PAGE System |
Application |
Comment |
---|---|---|
SDS-PAGE (Laemmli) |
Determination of subunit molecular weight Homogeneity test of a purified protein |
Native protein activity is lost Not suitable for low molecular weight proteins/peptides (<10 kDa) |
SDS-urea PAGE |
Separation of membrane proteins Suitable for low molecular weight proteins |
— |
Non-denaturing PAGE |
Homogeneity test of a purified protein |
Native protein activity usually retained Not reliable for molecular weight estimation |
Tricine PAGE |
Separation of low molecular weight proteins/peptides (1 – 40 kDa range) |
Protein band in the gel can be excised for amino acid sequencing without significant interference. |
Non-urea SDS-PAGE (modified Laemmli) |
Separation of low molecular weight proteins/peptides (as low as 5 kDa) |
— |
Acid-urea PAGE |
Separation of basic proteins such as histones |
Long run Proteins move toward cathode Electrode connection is opposite to the SDS-PAGE configuration |
CTAB-PAGE |
Determination of native molecular weight Native activity assay |
Proteins move toward cathode Electrode connection is opposite to the SDS-PAGE configuration |
Various polyacrylamide gel electrophoresis (PAGE) systems are known, and the choice of PAGE depends on the nature of the protein sample and the applications after electrophoresis (see Table 3.1).
Denaturing PAGE in the presence of sodium dodecyl sulfate (better known as SDS-PAGE) is a low-cost, reproducible, and rapid method for analyzing protein purity and for estimating protein molecular weight.1 SDS-PAGE is also employed for the following: (a) monitoring protein purification; (b) verification of protein concentration; (c) detection of proteolysis; (d) detection of protein modification; and (e) identification of immunoprecipitated proteins. SDS-PAGE can also be performed in a preparative mode to obtain sufficient protein for further studies. After electrophoresis the protein of interest is recovered from polyacrylamide by electroelution (see Section 3.2.1.7). The protein that is obtained by this process is generally used for raising antibodies or sequencing.
Figure 3.2 Denaturation of protein with sodium dodecyl sulfate, creating a highly negative charged molecule.
Mechanism
In SDS-PAGE, the sample applied to the electrophoresis has been treated with sodium dodecyl sulfate, an anionic detergent. This detergent denatures the proteins in the sample and binds strongly to the uncoiled molecule. Approximately one SDS molecule binds per two amino acids. The SDS molecules mask the surface charge of the native proteins and create a net negative charge resulting from the sulfate groups on the SDS molecule (Figure 3.2). Therefore, charge/size ratio is equal for all proteins, and separation can be achieved only on the basis of size. Low molecular weight proteins travel faster in the gel, and proteins of high molecular weight move slower in the gel. Because proteins are separated on the basis of size, their molecular weights can be estimated by running appropriate standard proteins of known molecular weights on the same gel.
The quality of the SDS is very important, because differential protein-binding properties of impurities such as C10, C14, and C16 alkyl sulfates can cause single proteins to form multiple bands in gels.
Electrophoresis can be performed in two buffer systems: continuous and discontinuous. A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel. The widely used SDS-PAGE, a modification by Laemmli 1 from those described in Ornstein 2 and Davis, 3 is a discontinuous system consisting of two contiguous but distinct gels: a resolving or separating (lower) gel and a stacking (upper) gel (Figure 3.3). The two gels are cast with different porosities, pH, and ionic strength. In addition, different mobile ions are used in the gel and electrode buffers.
Figure 3.3 Gel plate, showing the location of separating and stacking gels.
How Are Proteins Concentrated in the Stacking Gel?
The buffer discontinuity acts to concentrate large volume samples in the stacking gel, resulting in better resolution than is possible using the same sample volumes in gels without a stacking gel. Proteins, once concentrated in the stacking gel, are separated in the resolving gel. In SDS-PAGE, samples prepared in a low-conductivity buffer (0.06 M Tris-HCl, pH 6.8) are loaded between the higher conductivity electrode buffer (0.025 M Tris, 0.192 M glycine, pH 8.3) and the stacking gel buffer (0.125 M Tris-HCl, pH 6.8). When power is applied, a voltage drop develops across the sample solution, which drives the proteins into the stacking gel. During electro-phoresis, glycinate ions from the electrode buffer follow the proteins into the stacking gel. Between the highly mobile chloride ions in the front and the relatively slow glycinate ions in the rear, a high-voltage gradient forms. This causes SDS-protein complexes to form into a thin zone (stack) and migrate between the chloride and the glycinate ions. Most proteins usually move in the stacking gel (3 to 4%) due to large pore size, but at the interface of the stacking and resolving gels, the proteins experience a sharp increase in retardation due to the restrictive pore size of the resolving gel. In the resolving gel, the glycinate ions overtake the proteins, which continue to be slowed by the sieving of the matrix. Molecular sieving causes the SDS-polypeptide complexes to separate on the basis of their molecular weight. The molecular weight ranges of proteins that are separated depend on the percentage of the acrylamide gel (Table 3.2).
SDS-PAGE yields the molecular weight of the subunit that is non-covalently linked. To obtain the molecular weight of the subunit that is linked by disulfide bond, the presence of a reducing agent, such as 2-mercaptoethanol or dithiothreitol (DTT), is necessary in the sample buffer. The reducing agent breaks the disulfide bonds in the protein as follows:
Percentage (%) of Acrylamide Gel |
Separating Resolution (kDa) |
---|---|
15 |
15–45 |
12.5 |
15–60 |
10 |
18–75 |
7.5 |
30–120 |
5 |
60–212 |
Notes:
a Adapted from Reference 4 .
Of two gel formats (tube gel and slab gel), slab gels (formed between two sheets of supporting glass) are most widely used, since many samples can be run on the same gel, thereby providing uniformity during polymerization, staining, and destaining. Reagents for SDS-PAGE and electrophoresis cells of various designs are available from several vendors. For most analytical applications, the mini slab gel (8 × 10 cm) is generally used, due to the increased resolution and reduced amounts of time and materials needed for electrophoresis. The experimental procedures and reagents described below have been calculated for a mini gel system; however, working procedures for other systems are adapted easily.
Polyacrylamide gels with various pore sizes are made by varying the concentration of acrylamide. The choice of acrylamide concentration is determined by the molecular weight range of proteins to be separated (see Table 3.2). Recipes for making gels of varying concentration are shown in Table 3.3. Gels of fixed acrylamide concentrations are typically used on a daily basis because they are simple to prepare. When proteins of broad molecular weight range or higher resolution are desired, gradient polyacrylamide gels can be made (see Section 3.2.3). In practice, acrylamide solution (from the recipe in Table 3.3) is poured into a cassette made by joining two gel plates (usually made of glass) to form a separating gel (Figure 3.4 A). Spacers are placed between plates to make the cassette. Once the separating gel is polymerized, stacking gel is then made on top of the separating gel (Figure 3.4 B). Gels can be made in various thicknesses according to the thickness of the spacers (0.75 mm and 1.5 mm are common). Single or multiple gels can be made at a time. Gel casters of various sizes are commercially available for this purpose.
|
Final Acrylamide Concentration in the Separating Gel |
|||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Stock Solutions |
5 |
6 |
7 |
8 |
9 |
10 |
12 |
13 |
15 |
17 |
20 |
|
30% acrylamide/0.8% bisacrylamide |
2.50 |
3.00 |
3.50 |
4.00 |
4.50 |
5.00 |
6.00 |
6.50 |
7.50 |
8.5 |
10.00 |
|
4× separating gel buffer |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
|
Water |
8.75 |
8.25 |
7.75 |
7.25 |
6.75 |
6.25 |
5.25 |
4.75 |
3.75 |
2.75 |
1.25 |
|
10% ammonium persulfate |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
|
TEMED |
0.01 |
0.01 |
0.01 |
0.01 |
0.01 |
0.01 |
0.01 |
0.01 |
0.01 |
0.01 |
0.01 |
0.01 |
Figure 3.4 Preparation of gel and loading samples on the gel. A, Gel cassette; B, Pouring stacking gel on top of separating gel; C, Inserting comb into the stacking gel; and D, Loading samples into wells.
Working Procedure
Equipment
Reagents
Stock Solutions
Working Solutions
Procedure
Making the Gel Sandwich
Pouring the Separating Gel
Pouring the Stacking Gel
Once a gel has been made, the comb is removed from the gel. After washing the wells with the electrophoresis buffer, the gel sandwich is placed in the electrophoresis tank. The tank is filled with the electrophoresis buffer following the manufacturer’s instructions. Electrophoresis tanks from various commercial sources vary in size and shape. But, in all cases, the gel sandwich is submerged in the electrophoresis buffer and protein samples are loaded into wells through the buffer. Once samples are loaded, the electrophoresis lid is carefully closed; the chamber electrodes are attached to the power supply.
Working Procedure
Equipment
Reagents
Procedure
After electrophoresis, proteins are detected on the gel by using various stains (Coomassie blue, silver, Amido Black, etc.). Staining with Coomassie blue is rapid and the most common protein stain for routine work (Table 3.4). Compared to Coomassie stain, silver staining is a time-consuming, but more sensitive, method for staining proteins in gels. Silver staining should be used to assess the purity of a protein preparation, such as antigen preparation for development of polyclonal antibodies. Reversible stains such as Ponceau and India ink are generally used to visualize protein bands in gels prior to Western transfer or on membranes prior to protein elution (see Section 3.5). Protein staining with Procion blue can be used for quantification of protein in gels. 6 Many stains are commercially available (Table 3.5).
Coomassie Brilliant Blue dye (see Figure 2.6 A) is widely used to visualize proteins in polyacrylamide gels. 7 The staining is simple and can detect as little as 0.1 µg of protein in a single band. The dye binds primarily to positively charged amino acids, such as lysine and arginine. Thus, basic proteins tend to stain more strongly than acidic proteins. 8 The advantage of the Coomassie stain is that it is rapid and flexible. The gel can be stained in 5 to 10 min, followed by destaining that requires about 1 to 2 h. Coomassie blue turns the entire gel blue, and after destaining, the blue protein bands appear against a clear background. If staining appears to be incomplete after destaining, gel can be restained. If staining is not sensitive enough to detect all proteins, the gel can be rinsed and then subjected to the more sensitive silver staining procedure (see Section 3.2.1.3.2).
Stain |
Sensitivity |
Time Required |
Advantage |
Disadvantage |
Recommended Application |
---|---|---|---|---|---|
Coomassie |
100 ng |
5–10 min staining 1–3 h destaining |
Rapid Low cost After Coomassie stain, gel can be silver stained |
Accumulates large volume of methanol present in staining and destaining solution |
Routine work |
EZBlueTM (Sigma) |
2 ng |
5–10 min |
No fixation step |
Expensive |
Routine work |
Colloidal Coomassie |
10 ng |
5–10 min |
No destaining step |
Fixation required |
Routine work |
CBB in acid |
20 ng |
30–60 min |
No fixation step No destaining step |
Longer staining time |
Quantitation of protein in gel |
Silver (alkaline method) |
0.1 ng |
2 h |
Most sensitive, when sensitized with glutaraldehyde prior to staining |
Complex reagent preparation, unstable reagent |
To assess purity of protein preparation |
Silver (acid method) |
0.6–1.2 ng |
90 min |
Few steps Simple reagent preparation |
Less sensitive than alkaline silver stain method |
To assess purity of protein preparation |
Zinc |
5 ng |
25–40 min |
No fixation step Elution of unstained protein |
Multiple steps when toning reaction intended |
Peptide sequencing, antibody development |
Nile Red |
100 ng |
As little as 6 min |
Rapid |
UV light box and camera required for documentation |
Routine work |
Calconcarboxylic acid |
10 ng (during electrophoresis) 25 ng (post electrophoresis) |
30–70 min |
Migration of stained proteins during electrophoresis |
Simultaneous staining is less sensitive than post staining |
Routine work |
Eosin Y |
10 ng |
30 min |
Antigenicity of the stained protein retained |
Transilluminator required |
Antibody development |
Procion blue |
100 ng |
1.5 h staining 48 h destaining |
Proteins in gel can be quantitated |
Time consuming |
Quantitation of proteins |
Amido Black |
>100 ng |
2–4 h |
— |
Less sensitive |
— |
Fast Green FCF |
200 ng |
2–4 h |
— |
Less sensitive |
— |
Based on |
Stain |
Vendor |
---|---|---|
Coomassie Blue |
GelCodeR Blue Stain (Colloidal properties of Coomassie G-250) InstaStainTM Blue Gel Stain Paper (Coomassie dye in a solid phase) Coomassie Bio-Safe Coomassie EZ BlueTM Gel Stain |
Pierce
Pierce Bio-Rad Bio-Rad Sigma |
Silver |
GelCodeR Silver Stain GelCodeR SilverSNAPTM Stain Silver Stain (Meril) Silver Stain Plus (Gottlieb and Chavco) |
Pierce Pierce Bio-Rad Bio-Rad |
Zinc Reverse |
GelCodeR E-ZincTM Reversible Stain Zinc Stain |
Pierce Bio-Rad |
Fluorescent |
SYBR Red (Based on Nile Red) SYPROR Tangerine SYPROR Orange SYPROR Red |
Molecular Probes Molecular Probes Molecular Probes Molecular Probes |
Luminescent |
SYPROR Ruby Stain |
Molecular Probes |
Working Procedure
Equipment
Reagents
Coomassie Brilliant Blue R-250 | 1 g |
Methanol | 450 ml |
Water | 450 ml |
Glacial acetic acid | 100 ml |
Methanol | 100 ml |
Water | 800 ml |
Glacial acetic acid | 100 ml |
Procedure
Other Variations of Coomassie Stain
Coomassie Brilliant Blue G (0.04%, w/v) in 3.5% (w/v) perchloric acid can be used to stain proteins on SDS and non-denaturing polyacrylamide gels as well as agarose gels. 9 Proteins are stained in 30 to 60 min. No destaining step is required. Fixation step is also not required as perchloric acid can fix proteins during staining. However, SDS-PAGE gels are pre-fixed to remove SDS prior staining.
EZBlueTM Gel staining reagent (Sigma-Aldrich) is a one-step ultrasensitive stain based on Coomassie Brilliant Blue G-250. EZBlue stain can detect as little as 2 ng protein. It fixes proteins during staining, and thus a separate fixing step is not required. Destaining is also not required with this stain, although a water rinse after staining enhances sensitivity.
A colloidal concentrate of Coomassie Brilliant Blue G is available from Sigma-Aldrich. After dilution, the suspension contains 0.1% Brilliant Blue G, 0.29 M phosphoric acid, and 16% saturated ammonium sulfate. The stain is about tenfold more sensitive than the regular Coomassie stain. A fixation step is required prior to staining.
An advantage of these variations of the Coomassie stain is the absence of methanol, which is a regulated chemical waste.
Switzer et al. 10 introduced a silver stain, which is at least 100 times more sensitive than Coomassie stain. Several variations and modifications have been developed. However, silver staining is primarily achieved in two ways: an alkaline method based on the use of ammoniacal silver or silver diamine, and the use of silver nitrate in weakly acidic solution. Both procedures are based on the reduction of cationic silver to metallic silver. 11 Amino groups, especially the epsilon-amino group of lysine and sulfur residues of cysteine and methionine, are believed to react with silver cations. 12,13
Reaction
In the first method, ammoniacal silver or silver diamine is prepared by mixing silver nitrate with sodium hydroxide resulting in a precipitate of silver hydroxide, which is brought back into solution with the slow addition of ammonia as follows:
The gel is impregnated with silver diamine to allow the formation of a complex with the proteins. After removing the excess silver diamine from the gel, the complexed silver cations are reduced to metallic silver with formaldehyde in the presence of acid, usually citric acid.
The second method requires an initial gel soak in a weakly acidic silver nitrate solution and development with formaldehyde in the presence of alkali, usually sodium carbonate or sodium hydroxide. Sodium carbonate or other bases buffer the formic acid produced by the oxidation of formaldehyde, so that the silver ion reduction can continue until the protein bands appear in the gel.
Of the two methods, the alkaline silver nitrate method is more sensitive than the acidic silver nitrate method, but the former is more time consuming than the latter.
In some cases, prior to the silver nitrate step, gels are primed with a reducing agent like dithiothreitol or an oxidizing reagent like permanganate or dichromate. With Bio-Rad’s Silver Stain, the formation of a positive image is enhanced by dichromate oxidation, which may convert protein hydroxyl and sulfhydryl groups to aldehydes and thiosulfates, thereby altering the redox potential of the protein. Complexes formed between the proteins and dichromate may further form nucleation centers for silver reduction.
Among the various modifications, a method combining the use of glutaraldehyde treatment and the use of silver diamine to soak the gel was found to be most sensitive. 14 The increased sensitivity is probably due to increased reduction rate of silver on the proteins. 15
Before a protein gel can be stained, the proteins must be fixed, in order to minimize the diffusion of molecules in the gel. Fixation also elutes substances from the gel that may interfere with the establishment of the oxidation/reduction potential differences and with silver reduction. Ampholytes, detergents, reducing agents, initiators or catalysts, and buffer ion (glycine, chloride, etc.) must be removed. Water used in all silver stain reactions must be of 1 µmho conductance or less and free of organic contaminants.
Although silver staining normally produces a dark brown image, other colors may be produced when dense protein zones become saturated after prolonged development. Color production largely depends on the size and the distribution of the silver particles within the gel and the refractive index of the gel. 16
Working Procedure
Silver Diamine Method
Reagents
Procedure
All steps are performed at room temperature.
Acidic Silver Nitrate Method
Reagents
250 ml | ethanol |
60 ml | acetic acid |
0.25 ml | formaldehyde |
100 mg | silver nitrate |
70 µl | formaldehyde |
3 g | sodium carbonate (anhydrous) |
50 µl | formaldehyde |
1 mg | sodium thiosulfate (Na2S2O3.5H2O) |
(Note: all solutions should be made fresh except EDTA.)
Procedure
Procion blue MX-2G-125 dye can be used to quantitate proteins on gels (6). The lower detection limit is about 1 µg per band.
Reaction
Procion dye contains a dichlorotriazine group which reacts with hydroxyl and amino groups of proteins.
Working Procedure
Reagents
Procedure
Staining of proteins with Nile Red (9-diethylamine 5 H-benzo [α] phenoxazine-5-one) (Figure 3.5) is rapid. It detects as low as 0.1 µg protein/band.
Reaction
Figure 3.5 Diagram of Nile Red.
Nile Red is a fluorescent hydrophobic dye. It binds protein-SDS complexes. Since it also interacts with SDS micelles, SDS-PAGE is usually performed at a lower SDS concentration (0.05% instead of typical 0.1%) in order to reduce background. This concentration is lower than the critical micelle concentration.
Working Procedure
Equipment
Reagent
Nile Red: 50× concentrated stock (0.4 mg/ml) in dimethyl sulfoxide. Stable at room temperature for at least 3 months. Nile Red is commercially available from vendors such as Sigma (St. Louis, MO) and Eastman Kodak (Rochester, NY).
Ready-to-use Nile Red solution is available from Molecular Probes (Eugene, OR) under the trade name SYBR Red Protein Gel Stain.
Procedure
Unlike traditional staining methods such as Coomassie and silver stains, reverse staining methods stain the whole gel except the area of the protein bands. The sensitivity of zinc stain is comparable to Coomassie stain. Zinc reverse staining is achieved in three steps: (a) incubate the gel in sodium carbonate, (b) incubation in imidazole, and (c) finally incubation with zinc sulfate. No fixative solution is used in this method. Reverse stain is particularly useful when elution of unstained protein is intended for further analyses. Usually, the gel is kept in water after staining. However, a toning reaction with a mixture of potassium ferricyanide, o-tolidine, and sulfuric acid is necessary if gels should be dried.
Reaction
At alkaline pH, Zn2+ forms a white insoluble precipitate with imidazole. The white precipitate turns into a deep blue with toning reaction.
Working Procedure
Reagents
Procedure
Figure 3.6 Diagram of calconcarboxylic acid.
Calconcarboxylic acid [1-(2-hydroxy-4-sulfo-1-naphthylazo)-2-hydroxy-3-naphthoic acid, CNN] (Figure 3.6) can be used for simultaneous as well as post-electrophoretic protein staining. 17 For simultaneous staining of proteins during electro-phoresis, CNN is simply added in the upper reservoir. The sensitivity of this stain is about 10 ng and 25 ng by post-staining and simultaneous staining, respectively.
Reaction
Staining of proteins with CNN is pH dependent (intense staining at pH 1.6 to 4.4 and weak at alkaline pH). At acidic pH, various functional groups of CNN (carboxyl, sulfonic acid, hydroxyl) probably form electrostatic bonds with protonated amino groups in proteins. The lower staining intensity in simultaneous staining is probably due to alkaline pH of the electrophoresis buffer.
Working Procedure
Reagents
Procedure for Simultaneous Staining
Procedure for Post-electrophoretic Staining
The Eosin Y staining method detects proteins on gels as well as on membranes more rapidly than most Coomassie and silver staining methods. The stain can detect as little as 10 ng of protein. An advantage of this stain is that the antigenicity of the stained protein is retained.
Reaction
Protein staining may occur by means of hydrophobic interaction between aromatic rings of eosin Y and the protein and by hydrogen bonding between hydroxyl groups of eosin Y and the protein.
Working Procedure
Equipment
Reagents
Procedure
Amido Black (also known as Naphthol Blue Black, Acid Black 1, or Buffalo Black NBR) can be used to stain proteins on gels. The detection sensitivity is lower than that of Coomassie Blue. Fixation is recommended for this stain.
Working Procedure
Reagents
Procedure
Fast Green FCF dye is used for protein staining in SDS-PAGE, native PAGE, and isoelectric focusing gels. 18 After electrophoresis, fixing is required for maximum sensitivity. Sensitivity is about two times less than Coomassie staining.
Working Procedure
Reagents
Procedure
Vendors of several commercial protein stains (Molecular Probes, Bio-Rad, Pierce, Sigma) offer ready-to-use convenient packs (Table 3.5). The sensitivity of some of these is comparable to silver stain. For example, Molecular Probes’ SYPROR Tangerine, SYPROR Orange, and SYPROR Red are fluorescent-based stains (Ex/Em wave lengths are 490/640, 470/570, and 550/630, respectively) and can detect as little as 4 ng protein per band. SYPROR Ruby stain (Molecular Probes) is an ultrasensitive luminescent stain for the detection of proteins on polyacrylamide gels (lower detection limit 75 fmol).
Subunit molecular weight of a protein is usually determined on SDS-PAGE, since the migration of protein is proportional to the mass. A standard curve is generated from proteins of known molecular weight (known as standard proteins), and the molecular weight of unknown protein is determined from the curve. The standard curve is obtained by plotting the relative mobility (Rf) value (in x-axis) and log10 of the molecular weight (in y-axis). Rf value is determined as follows:
Following electrophoresis and staining, the migration of proteins and tracking dye (bromophenol blue) can be measured.
Following staining of proteins in gels, individual protein bands can be quantitated by densitometric scan over a limited range of protein concentration (1 to 10 µg/band). This technique clearly provides an advantage over the estimation of crude proteins (mixture of proteins) in solution where quantitation of individual proteins cannot be obtained. For densitometric quantitations, the most suitable protein stains are Procion blue stain, 6 zinc stain, 19 and colloidal Coomassie stain. 20 Staining of proteins with these procedures is discussed in Section 3.2.1.3. A standard curve is drawn from known amounts of proteins, and the amount of the unknown protein is then determined from the plot.
Alternatively, protein quantitation is achieved by eluting dye from the stained protein bands. 21
For long-term preservation, stained gels can be dried on thick paper backing under vacuum 22 or between sheets of cellophane at atmospheric pressure. 23 Gels dried between transparent sheets are useful for densitometry.
Working Procedure for Vacuum Drying
Materials
Equipment
Procedure
Working Procedure for Air-Drying
Materials
Cellophane
Equipment
Gel drying frame (Figure 3.7)
Procedure
Figure 3.7 Drying gel using gel drying frame. The frame in figure is available from Diversified Biotech (Boston, MA).
Proteins from acrylamide gels can be extracted by electroelution 24 or protein diffusion. 25 For this purpose, stained gels (usually Coomassie or zinc reverse stained) containing protein bands are cut out with a razor blade, minced, and subjected to elution of proteins employing either method. Various electroelution devices are commercially available (from vendors such as Bio-Rad and Millipore) and should be operated following the manufacturer’s instructions.
For extraction of proteins by diffusion, an appropriate buffer is added to the minced gel slice, incubated for 15 min to several hours, and centrifuged, and the supernatant is collected. Ball 21 described an efficient and simple procedure to isolate Coomassie stained protein from gel slices. In this procedure, the gel slice is incubated with 1 ml of 3% SDS in 50% isopropanol at 37°C for 24 h, and after centrifugation supernatant is collected.
SDS-urea PAGE is often used for proteins of low molecular weight and membrane proteins. 26,27 In SDS-PAGE, the migration of low molecular weight proteins may not be proportional to their molecular weight, as the protein charge properties become significant relative to the mass. SDS-urea PAGE is suitable for membrane proteins, as they may not be soluble at conditions used in SDS-PAGE.
Working Procedure
All procedures for SDS-urea PAGE are essentially similar to those described for SDS-PAGE except the composition of gels (both separating and stacking) and sample loading buffer. These should contain 8 M urea. The recipe for 10% separating gel is shown below as an example.
For 10 ml 10% Separating Gel
Acrylamide stock solution (see Section 3.2.1.1) |
3.3 ml |
4× separating gel buffer (see Section 3.2.1.1) |
2.5 ml |
Urea |
4.8 g (equivalent to 3.6 ml) |
Water |
0.6 ml |
10% ammonium persulfate |
50 µl |
TEMED |
5 µl |
For 4 ml 5% Stacking Gel
Acrylamide stock solution (see Section 3.2.1.1) |
0.67 ml |
4× stacking gel buffer (see Section 3.2.1.1) |
1.0 ml |
Urea |
1.9 g |
Water |
0.93 ml |
10% ammonium persulfate |
30 µl |
TEMED |
5 µl |
Although polyacrylamide gels of fixed concentrations are widely used for routine analyses, the use of gradient polyacrylamide gels (increasing acrylamide concentration and hence decreasing pore size) has at least two advantages over fixed-concentration acrylamide gels. First, a gradient gel allows the separation of proteins of a larger range of molecular weights compared to a fixed-percentage gel. The second advantage of the gradient gels over the fixed-percentage gels is that the proteins of very close molecular weights can be resolved as sharp bands. However, the gradient gel requires additional equipment (such as gradient maker, pump, and tubing) and special attention when pouring the gel mixture into the gel sandwich. Air bubbles lodged in the tubing or in the gradient maker can cause the gradient to form unevenly. Fortunately, precast gradient gels are commercially available from Pharmacia, Bio-Rad, Jule Inc., and other manufacturers. The two common ranges of gradient gels are 3 to 30% and 5 to 20%, which resolve 13 to 950 kDa and 15 to 200 kDa, respectively.
Mechanism
In gradient gels, proteins of a wide molecular weight range enter the gel. Proteins of high molecular weight start to resolve immediately according to the pore size of the gel. Proteins of low molecular weight migrate freely in the beginning of the gel and start to resolve when they reach the appropriate percentage of gel with the smaller pore size. Proteins travel until they reach critical pore size (pore limit), which impedes further progress. At this point, the pattern of protein bands does not change significantly with time, although migration does not stop completely. 28
Regarding the separation of two proteins of very close molecular weights, each protein travels through the gel until it reaches its pore size limit. At this point, the protein stacks up, as the gel pore is too small to allow further migration of protein. A similar protein but with slightly lower molecular weight is able to travel further before it reaches its pore size limit and stacks as a sharp band.
The following procedure shows the preparation of two 0.75-mm-thick gradient gels. The amounts of each component can be scaled up when multiple gels are to be prepared. However, it is important to assemble all gel sandwiches in a single gel caster.
Working Procedure
Equipment
Reagents
Procedure
Figure 3.8 Diagram of gradient maker.
5% (light solution) | 20% (heavy solution) | |
30% acrylamide stock solution | 1.67 | 6.67 |
4× stacking gel buffer | 2.5 | 2.5 |
Water | 5.8 | 0 |
Sucrose | 0 | 1.5 g |
(equivalent to 0.8 ml) | ||
10% ammonium persulfate | 50 µl | 50 µl |
TEMED (To be added later) | 5 µl | 5 µl |
Non-denaturing PAGE, also called native PAGE, refers to the electrophoretic separation of the native protein. This can be performed following the standard Laemmli SDS-PAGE protocol described above, except the solutions contain no SDS or reducing agent. In non-denaturing PAGE, separation of proteins depends on many factors such as size, shape, and native charge. Native PAGE is mostly used to determine the homogeneity of the purified protein. Native PAGE is very useful to visualize enzyme or lectin activity after electrophoretic separation. Unlike SDS-PAGE, in which the denatured proteins are uniformly negatively charged and their mobilities are dependent on their molecular weights, determination of the native molecular weight using native PAGE is not reliable, as the mobility of the native proteins depends on both molecular weight and charge.
This difficulty is partly overcome by operating native PAGE at a high pH buffer (pH 8.8). At this pH, most proteins are negatively charged and thus move toward the anode. In order to determine molecular weight using non-denaturing gel electrophoresis, the protein should be run under a variety of acrylamide concentration (usually 4 to 12%). The results from these conditions are used to adjust the effect due to protein charge. In native PAGE, acrylamide concentration may vary from 5 to 15% and acrylamide:bisacrylamide ratio may vary from 20:1 to 50:1 to achieve different sieving effects. The ionic strength is an important factor in the native PAGE, especially when the protein’s activity is to be investigated after electrophoresis. High ionic strength generates heat during electrophoresis, resulting in a loss of protein activity. However, if the ionic strength is too low, proteins may aggregate non-specifically. Typically, ionic strength is kept in the range of 10 to 100 mM. All steps are usually performed at 0 to 4°C to minimize the loss of protein activity by denaturation and to reduce proteolysis.
Native PAGE is performed in two ways: (a) discontinuous: both stacking and separating gels like SDS-PAGE, and (b) continuous: no stacking gel. Continuous gel electrophoresis is simpler than discontinuous, as no stacking gel is involved. However, the lack of stacking gel often results in diffused or poorly resolved bands. In continuous native PAGE, ionic strength of the protein buffer is kept five- to tenfold lower than the gel buffer in order to obtain the sharpest bands. The volume of the protein sample is kept as small as possible. Thus, the protein concentration should be high (2 to 10 mg/ml). Buffers for continuous native PAGE may be the same as described below except that those pertaining to the stacking gel are omitted. Additional buffers are described elsewhere. 29 The procedure for discontinuous non-denaturing gel electrophoresis is described below.
Working Procedure
Equipment
Reagents
Stock Solutions
Working Solutions
Preparation of Gel
The recipe for making gels of varying strengths is essentially identical to the amounts shown in Table 3.3. Preparation of gel cassettes, pouring separating and stacking gel into the gel cassettes, and running gels are similar to denaturing PAGE.
Tricine PAGE is mainly used for the separation of low molecular weight peptides (range 40 to 1 kDa), 30 which cannot be resolved in Laemmli SDS-PAGE. In the Laemmli system, SDS and smaller proteins comigrate and thus obscure the resolution. In Tricine (N-Tris [hydroxymethyl] methylglycine) gel electrophoresis, Tricine separates SDS and peptides, thus improving resolution. The Tricine PAGE system has an additional advantage. Since glycine (which interferes with the amino acid sequence analyses) is replaced by Tricine in the electrophoresis buffer, protein bands in the gel can be excised for amino acid sequencing.
Working Procedure
Reagents
Procedure
Separating gel | Stacking gel | |
30% acrylamide stock solution | 15.0 ml | 1.62 ml |
Separating/stacking gel buffer | 10.0 ml | 3.10 ml |
Water | 1.83 ml | 7.78 ml |
Glycerol | 3.17 ml | |
10% ammonium persulfate (to be added later) | 50 µl | 50 µl |
TEMED (to be added later) | 5 µl | 5 µl |
Okajima et al. 31 described a modification of Laemmli SDS-PAGE for separation of peptides as low as 5 kDa. In this modification, the concentration of buffers is increased to provide better separation between the stacked peptides and the SDS micelles.
Working Procedure
Reagents
Procedure
Separating gel | Stacking gel | |
30% acrylamide stock solution | 10.0 ml | 0.65 ml |
Separating gel buffer | 3.75 ml | — |
Stacking gel buffer | 1.25 ml | |
10% SDS | 0.15 ml | 50 µl |
Water | 1.0 ml | 3.0 ml |
10% ammonium persulfate (to be added later) | 50 µl | 25 µl |
TEMED (to be added later) | 10 µl | 5 µl |
Panyim and Chalkley 32 introduced a continuous acetic acid urea PAGE for the separation of unmodified histone from its monoacetylated or monophosphorylated form. The procedure can separate similar basic proteins based on differences in effective charge as well as differences in size. Proteins of a slightly different charge such as unmodified and acidic acetylated derivative can be separated in acid urea PAGE. Urea is commonly used in this PAGE, mainly to disrupt any aggregation and to increase the density of the loading solution. In this system, riboflavin or riboflavin 5′-phosphate is used as initiator of photo-polymerization of acrylamide, as ammonium persulfate interferes with stacking of the proteins in the gel. Chloride ions also interfere with the stacking system. Thus, protein samples and glycine used in the elctrophoresis buffer should be free of chloride salts.
Mechanism
In acid urea PAGE, the samples are electrophoresed in acetic acid buffer (pH around 3). At this pH, basic proteins with a high isoelectric point get positively charged and move toward the cathode under an electric field.33
Working Procedure
Reagents
Procedure
Final concentration | ||
Acrylamide stock solution | 17.5 ml | 15% |
Bisacrylamide stock solution | 2.8 ml | 0.1% |
Glacial acetic acid | 4.2 ml | 1.0 M |
Concentrated ammonium hydroxide | 0.23 ml | 0.05 M |
Urea | 9.6 g | 8.0 M |
Water to make 18.6 ml | ||
Riboflavin 5'-phosphate (to be added later) | 1.3 ml | 0.0004% |
TEMED (to be added later) | 0.1 ml | 0.5% |
Final concentration | ||
Acrylamide stock solution | 1.34 ml | 4% |
Bisacrylamide stock solution | 1.28 ml | 0.16% |
Glacial acetic acid | 1.14 ml | 1.0 M |
Concentrated ammonium hydroxide | 0.07 ml | 0.05 M |
Urea | 9.6 g | 8.0 M |
Water to make 18.6 ml | ||
Riboflavin 5′-phosphate (to be added later) | 1.3 ml | 0.0004% |
TEMED (to be added later) | 0.1 ml | 0.5% |
Bonner et al. 34 described acid urea PAGE system in the presence of non-ionic detergent Triton X-100 for separation of core histones. This is based on the observation that core histones but not linker histones or any other protein bind Triton. 35
Although Laemmli PAGE is widely used for testing the purity of a protein and the determination of its subunit size, this procedure is not suitable to assess the biological activity of proteins treated with SDS. Cetyltrimethylammonium bromide (CTAB) PAGE allows the sample solubilization in CTAB and molecular size-dependent separation of proteins in an arginine/Tricine buffer, with the retention of native activity. 36,37 The following working procedure is based on Akins et al., 37 who described CTAB PAGE in a discontinuous gel format.
Mechanism
In the CTAB PAGE system, proteins get positively charged and thus migrate toward the cathode under electric field. The arginine in the electrophoresis buffer also migrates toward the cathode, as arginine is positively charged at the electrophoresis buffer pH 8.2 (pI of arginine is 10.8). However, at the stacking gel (pH 9.96) arginine will have a lower net positive charge and will move slowly. In the interface zone between the upper tank buffer and the stacking gel/sample buffer, sodium ions (Tricine-NaOH) move ahead of the slow-moving arginine. The CTAB-coated proteins migrate more quickly in this interface zone than in the sodium-containing zone and “stack” as the interface advances.
Working Procedure
Reagents
Procedure
40% acrylamide stock solution | 2.5 ml |
Separating gel buffer | 2.5 ml |
Distilled water | 4.89 ml |
10% Ammonium persulfate (to be added later) | 0.1 ml |
TEMED (to be added later) | 0.01 ml |
40% acrylamide stock solution | 1.0 ml |
Stacking gel buffer | 2.5 ml |
Distilled water | 6.39 ml |
10% ammonium persulfate (to be added later) | 0.1 ml |
TEMED (to be added later) | 0.01 ml |
Isoelectric focusing (IEF) is an electrophoretic method in which amphoteric molecules are separated as they migrate through a pH gradient. Polyacrylamide gels are generally used for focusing proteins. However, for proteins larger than 200,000 dalton (Da), 1% agarose gels can be employed.
Mechanism
The net charge on a protein is the algebraic sum of all its positive and negative charges. At physiological pH, lysine, arginine, and histidine residues in a protein are positively charged, while aspartic acid and glutamic acid carry a negative charge. So the net charge of a protein at a specific pH depends on the relative number of positive and negative charges. The pH at which a protein carries no net charge (total positive charge equal to total negative charge) is called its isoelectric point (pI). Below the pI the protein carries a positive charge, and a negative charge at pHs above pI. When protein is placed in a medium with varying pH and subjected to an electric field, it will initially move toward the electrode with the opposite charge. During migration through the pH gradient, the protein will either pick up or lose protons. As it does, its net charge and mobility will decrease, and at its pI the protein will stop moving. This type of motion is in contrast to conventional electrophoresis, in which proteins continue to move through the medium until the electric field is removed. In, proteins migrate to their steady-state positions from anywhere in the system, and thus the location of sample application is arbitrary.
The key to IEF is the establishment of stable pH gradients in electric fields. This is most commonly accomplished by means of commercially available, synthetic carrier ampholytes (amphoteric electrolytes). These compounds are mixtures of relatively small (600 to 900 Da), multicharged, amphoteric molecules with closely spaced pI values and high conductivity. 39 Under the influence of an electric field, carrier ampholytes partition themselves into smooth pH gradients, which increase monotonically from the anode to the cathode. The slope of the pH gradient is determined by the pH interval covered by the carrier ampholyte mixture and the distance between the electrodes. Isoelectric focusing is usually carried out in a denaturing gel system with urea. Charged denaturing agents such as SDS and sodium deoxycholate should not be used, as these interfere with the electrophoresis. Iso-electric focusing can also be carried out in a non-denaturing system, when functions of proteins (e.g., enzyme activity, lectin activity) are studied after focusing. Table 3.6 shows some common problems in isoelectric focusing and their remedies.
It is important to perform isoelectric focusing on a device where efficient gel cooling is achieved. This is required to maintain high-voltage gradient for better resolution of protein bands. Isoelectric focusing can be performed on either slab gels or tube gels. Several devices are commercially available for running slab gels (vertical and horizontal) and tube gels. A procedure for isoelectric focusing gel electrophoresis on a vertical slab gel format is described here.
Working Procedure
Equipment
Reagents
Problem |
Cause |
Remedy |
---|---|---|
High background staining |
Incomplete removal of ampholytes from the gel |
Increase time of fixing with 1% TCA |
Wavy bands |
High salt content in the sample Impurities in the ampholyte or electrolyte solutions Dirty electrodes |
Dialyze the sample in low salt buffer Use fresh ampholytes and electrolytes Clean electrodes |
Streaking bands |
Protein aggregation or precipitation Presence of nucleic acids in the sample Modification of protein may occur such as oxidation of cysteine, deamination of asparagine and glutamine, carbamoylation of protein by isocyanate present in impure urea. |
Centrifuge samples before loading Remove nucleic acids Remove isocyanate impurities by prerunning the gel. Handle sample properly to avoid other modification. |
Overlapping bands |
Complex protein mixtures |
Change the pH range of the gel |
Skewed bands |
Faults in the pH gradient |
Verify that the electrodes are clean for good contact |
Fuzzy bands |
Incomplete focusing Large proteins and thus restricted mobility |
Increase voltage gradient incrementally towards the end of the run Use more porous agarose gel for large proteins |
Missing or faint bands |
Proteins have not been denatured during fixation |
Increase TCA concentration |
Uneven pH gradient |
Electrode contact is not parallel to the gel Impurities within the gel Ampholyte concentrations are too low |
Make sure electrodes are parallel to the gel Use reagents of highest grade Increase ampholyte concentrations |
Notes:
a Based on Reference 40.
Gels with pH Range |
Ratio of Ampholytes |
% in Final Gel Solution |
---|---|---|
pH 3.5–10 |
pH 3.5–10 |
2.4 |
pH 4–6 |
pH 3.5–10 |
0.4 |
|
pH 4–6 |
2.0 |
pH 6–9 |
pH 3.5–10 |
0.4 |
|
pH 6–8 |
1.0 |
|
pH 7–9 |
1.0 |
pH 9–11 |
pH 3.5–10 |
0.4 |
|
pH 9–11 |
2.0 |
Notes:
a Based on Reference 41.
Procedure
Preparation of Gels
Acrylamide stock solution (see Section 3.2.1.1) | 2.0 ml |
Water | 5.4 ml |
Ampholyte solution, pH 3.5-10 | 48 µl |
Ampholyte solution, pH 4-6 | 240 µl |
Urea, ultrapure | 6 g |
Set Up Gel
Sample Preparation and Loading
Final concentration | ||
Urea, ultrapure | 6 g | 8 M |
Ampholyte solution, pH 3.5 to 10 | 20 µl | |
Ampholyte solution, pH 4 to 6 | 100 µl | |
20% Triton X-100 | 500 µl | 2% |
2-mercaptoethanol | 5 µl | 1% |
1% bromophenol blue | 200 µ | |
Distilled water | 1.7 ml |
Running Gel
Cutting Gel Slices for pH Determination
Fixing and Staining the Gel
Native Isoelctric Focusing Gel
The working procedure for native isoelectric focusing is essentially identical to denaturing isoelectric focusing except some modifications in the recipe of the gel solution and the gel-loading buffer.
Recipe for Native Isoelectric Focusing Gel
Acrylamide stock solution (see Section 3.2.1.1) |
2.0 ml |
Water |
9.7 ml |
Ampholyte solution, pH 3.5 to 10 |
48 µl |
Ampholyte solution, pH 4 to 6 |
240 µl |
10% ammonium persulfate (to be added later) |
50 µl |
TEMED (to be added later) |
20 µl |
Recipe for Native Gel Sample Buffer (2 × )
Glycerol |
3.0 ml |
Ampholyte solution, pH 3.5 to 10 |
33.3 µl |
Ampholyte solution, pH 4 to 6 |
166.7 µl |
Distilled water |
1.8 ml |
Isoelectric Focusing in Horizontal Slab Gel
Isoelectric focusing in a horizontal slab gel format has several advantages over the vertical format. Cooling of the gel during electrophoresis is very efficient, as the gel remains flat on the cooling plate. Only a few milliliters of electrode solutions (enough to soak electrode strips) are required for electrophoresis. The gel size can be adjusted as needed. Sample can be added at any position in the pH gradient. Larger sample volume (more than 10 to 15 µl) can be added on a slab gel. Precast isoelectric focusing gels (polyacrylamide gel on a plastic support film) with varying pH ranges are commercially available (Amersham Pharmacia). Electrophoresis should be performed according to the manufacturer’s instructions.
In 2D gel electrophoresis, protein separates in two dimensions: the first dimension on the basis of pI and the second dimension based on subunit molecular weight. 44 Usually, isoelectric focusing is performed on a tube gel of very small diameter or on a thin gel strip, and after completion of the run the gel is placed horizontally onto the top of a polymerized slab gel for SDS-PAGE. In this system proteins are separated into many more components than is possible with conventional one-dimensional electrophoresis (Figure 3.9). Due to greater resolution, it is possible to quantitate differentially expressed proteins during a certain biological process. The powerful technique of protein separation and identification under the heading “Proteomics” is based on the principle of 2D electrophoresis. Procedures for 2D gel electrophoresis can be adopted from the isoelectric focusing and SDS-PAGE procedures described above. Additionally, 2D equipment and procedures for 2D electro-phoresis are available from various commercial sources such as Hoefer, Bio-Rad, and Millipore.
Western blotting refers to the electrophoretic transfer of the resolved proteins or glycoproteins from a polyacrylamide gel to a membrane such as nitrocellulose and polyvinylidene difluoride (PVDF). 45 The immobilization of proteins on a membrane is more useful than working on the gel for a number of reasons: (a) proteins are more accessible, (b) membranes are easier to handle than gels, (c) smaller amounts of reagents are needed, and (d) processing time is shorter. 46 Following the transfer of proteins to a membrane, a wide variety of applications can be carried out on the immobilized proteins such as immunodetection (see Chapter 6), carbohydrate detection (in case of glycoprotein see Chapter 7), and amino acid analysis and protein sequencing. Other applications involved in immobilized proteins are (a) epitope mapping, (b) ligand binding, (c) cutting out protein band for antibody production, and (d) structural domain analysis (see Figure 3.10). In most applications, immobilized proteins or glycoproteins can be identified and visualized by using very specific and sensitive detection techniques (immunological or biochemical). For example, as low as 1 to 10 pg of protein can be detected employing immunological techniques. Because of its wide applications and flexibility in protein detection methods, Western blotting has become a very popular and convenient method for analysis of denatured proteins.
Figure 3.9 2D gel of an aqueous extract of E. coli stained with Coomassie Blue. Isoelectric focusing in the first dimension was performed in Protein cell (Bio-Rad, Hercules, CA) on IPG strips in the pH range 3 to 10. SDS-PAGE in the second dimension was performed on a 4 to 15% gradient gel (Bio-Rad). Protein markers are shown in the left (indicated by arrow).
Figure 3.10 Flow diagram showing applications of immobilized proteins.
Mechanism
Proteins are transferred from the SDS-PAGE gels, in which all proteins are negatively charged due to the SDS treatment. In an electric field, these negatively charge proteins migrate towards the positive and get immobilized on the membrane.
Protein transfer is usually accomplished by one of two electrophoretic methods: semi-dry blotting and wet blotting. In the former method, the gel and immobilizing membrane matrix are sandwiched between buffer-wetted filter papers, and a current is applied for 10 to 30 min. In wet blotting, the gel-membrane matrix sandwich is submerged in a transfer buffer and current is applied for 45 min to overnight. Due to its greater flexibility, wet blotting will be described here.
Blotting Membrane
Proteins in acrylamide gels can be transferred to nitrocellulose, PVDF, nylon, or carboxymethyl cellulose. However, for most applications, nitrocellulose and PVDF are preferred for the following reasons (see also Table 3.8). Nitrocellulose is relatively inexpensive, and its non-specific binding to the antibody can largely be blocked. PVDF is more expensive than nitrocellulose, but is ideal for N-terminal amino acid sequencing and amino acid analysis, since the membrane is resistant to acid and organic solvents. In contrast, blocking of the non-specific protein band in nylon is cumbersome because of the high-charge density of the matrices. Protein staining in nylon with common anionic dyes (Coomassie Blue, Amido Black, etc.) is not possible due to the positive charge of the nylon matrix. However, nylon is used when (a) higher protein binding is required, (b) a protein binds weakly to nitrocellulose (especially high molecular weight), and (c) greater resistance to mechanical stress is desired. The binding capacity of nylon is almost eightfold more than that of nitrocellulose (80 µg/cm2). 46
The efficiency of Western transfer depends on several factors such as composition of buffer, time, voltage, and size of the protein; percent acrylamide; and the thickness of the gel. An optimization is required for each protein, and the efficiency of transfer can be assessed by staining the blots with any blot stain (see Section 3.5.1). In general, proteins of low molecular weight transfer more easily than those of high molecular weight. Proteins transfer more effectively from low-percent acrylamide gels than from high-percent gels. Methanol improves protein binding to nitrocellulose membrane, but inhibits transfer. SDS is sometimes added to the transfer buffer to improve transfer of large proteins, but unfortunately it inhibits protein binding to the membrane. Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) is commonly used for most proteins. When the transferred protein is used for amino acid analyses, the Towbin buffer is replaced by the CAPS buffer (10 mM 3-(cyclohexylamino)-1-propanesulfonic acid, 10% methanol, pH 11.0) to avoid interference with the analyses due to the presence of glycine. When protein is transferred from acid urea gel or isoelectric focusing gel, acetic acid (0.7%) is used as a transfer buffer. The apparatus for Western transfer is available from several vendors such as Hoefer, Bio-Rad, and Pharmacia and can easily be operated following the manufacturer’s instructions. A procedure for Western transfer using Hoefer apparatus is described below.
Membrane |
Commercial Source |
Charge of the Membrane |
Nature of Interaction between Membrane and Protein |
Remarks |
Recommended Applications of the Transferred Protein |
---|---|---|---|---|---|
Nitrocellulose |
Bio-Rad |
Negative |
Hydrophobic and electrostatic forces |
Low cost Mechanically fragile |
Immuno detection |
PVDF |
Millipore |
Negative |
Hydrophobic forces |
High cost |
N-terminal sequencing, amino acid analysis, immunodetection, ligand binding |
Nylon |
Millipore |
Positive |
Electrostatic forces |
Mechanically strong |
Multiple reprobing |
Carboxymethyl cellulose |
Millipore |
Negative |
Ionic interactions |
Very high capacity for histones Elution step is required before sequencing |
Sequencing of basic proteins |
Working Procedure
Equipment
Reagents
Procedure
Figure 3.11 Preparation of sandwich for Western transfer.
Membrane-immobilized proteins are often visualized to monitor the efficiency of transfer prior to further processing. Several stains are used for blot membranes, but anionic dyes such as Amido Black or India ink are less satisfactory for nylon membranes.
Staining with Coomassie Brilliant Blue
Immobilized proteins can be visualized in a few minutes with Coomassie staining. As this staining is irreversible, blot membranes that are subject for immunodetection should not be stained with Coomassie. However, one important application of Coomassie blue is to stain a portion of the membrane and match the stained proteins with the immunodetected proteins.
Working Procedure
Reagents
Staining Procedure
Staining with Amido Black
A protein band of lower microgram range can be detected with Amido Black satin.47
Working Procedure
Reagents
Procedure
Staining with India Ink
With this staining, protein bands appear as black on a gray background. 48 The stained membrane can be stored for at least one month without any loss of sensitivity.
Working Procedure
Reagents
Procedure
Staining with Ponceau S
Staining of immobilized proteins with Ponceau S is a reversible procedure, since the stain can be washed off completely with water. This stain is not very sensitive. Nonetheless, it is often used to monitor the transfer of protein prior to immunode-tection or other applications. The stain is also used to identify bands for micro sequencing.
Working Procedure
Other Stains for Blot Membranes
Several stains for blot membranes are commercially available. They are usually more sensitive than Ponceau or Amido Black. In most cases, the identities of the staining reagents are trade secrets. The staining procedures are available from vendors. MemCodeTM Reversible Protein Stain (Pierce, Rockford, IL) is used to stain the protein band on nitrocellulose membranes. The stain on blots can be washed off quickly for immunodetection or other applications. This stain is not suitable for PVDF membrane. SYPROR Ruby protein blot stain (Molecular Probes, Eugene, OR) is a very sensitive reagent to detect proteins on both nitrocellulose and PVDF membranes.
Recovery of proteins from membranes is often needed for many applications such as amino acid composition analysis, for protein sequencing, and as an immunogen. Several solvents can be used to elute protein from the membrane, and the choice of the solvent system depends on the intended application. For example, acetonitrile or n-propanol usually maintains the protein structure and thus can be used as an immunogen or antigen in radioimmunoassays. Detergent-based systems are used to elute proteins when proteolytic and analytical manipulations are desired. Detergent elution is more effective than elution with organic solvents.
Working Procedure
Elution with an Organic Solvent System
Elution with a Detergent-Based Solvent System
In this procedure molecules such as proteins, glycoproteins, peptides, and DNA are separated in a capillary tube (usually made of silica, 10 to 100 µm diameter) under a potential difference produced at two ends. The most common type of capillary electrophoresis is capillary zone electrophoresis (CZE), which relies on simple instrumentation consisting of a capillary column, a detector, and a high-voltage power supply (Figure 3.12). The two ends of the capillary tube are immersed in reservoirs containing electrolytes, which serve as electrodes. These electrodes are connected to a high-voltage power supply. A sample is introduced at one end of the capillary (inlet), and upon applying an electric field, sample components are separated as they travel through the capillary toward the other end (outlet). At the far end of the capillary, the separated components are sensed by a detector, and output signal is recorded. Since the walls of the capillary have a standing charge, an electroosmotic flow of water is produced from anode to cathode (Figure 3.13). So migration of a positively charged molecule from anode to cathode depends on the applied voltage gradient and electroosmotic flow. Uncharged molecules are separated on a silica capillary because of the electroendo-osmotic flow. For charged molecules, the apparent rate of migration is the algebraic sum of electrophoretic mobility and electroosmotic flow. Electrophoretic mobility is dependent on the mass/charge ratio.
Figure 3.12 Schematic representation of capillary electrophoresis apparatus.
Figure 3.13 Separation of sample in the silica capillary.
Figure 3.14 Silanol equilibrium.
The silica capillary columns are usually coated to reduce electroosmotic flow, resulting in improved separation. In the presence of electroosmotic flow, charged molecules migrate in an eliptical shape, but when migration is solely by the applied voltage gradient, the molecule front is plug shaped, resulting in a sharp peak.
The use of a buffer at extreme pH (high about pH 10 and low at about 2) results in a decrease in electrostatic adsorption. The silanol group is negatively charged at high pH and is protonated at acidic pH (Figure 3.14). At pH 10, most proteins (except very basic proteins) are negatively charged, and since the capillary wall is also negatively charged, the electrostatic interaction is minimized. Similarly at very low pH (about 2) both capillary wall and proteins are positively charged, resulting in a reduced electrostatic adsorption of proteins onto capillary wall. However, this practice is not popular because of possible denaturation and the loss of biological activities of proteins at extreme pH.
Detection of Protein
In contrast to standard liquid chromatography, where proteins are usually detected at 280 nm (the path length of the absorbance detector is usually 1 cm), detector signal in a capillary electrophoresis system is not satisfactory due to a very short detection path length (25 to 75 µm). Although the absorbance of protein at 200 nm is about 50- to 100-fold greater than that at 280 nm, detection in the low UV region is also not suitable for many applications in capillary electrophoresis. Alternatively, proteins can be detected by the intrinsic fluorescence of their tryptophan and tyrosine residues. However, the detection of intrinsic protein fluorescence requires very costly laser detectors (49). Thus, for capillary electrophoresis, pre- and postcolumn derivatization techniques have been developed to increase detection sensitivity of proteins.
Detection Using Precolumn Derivatization of Proteins
Precolumn derivatization is widely used for analysis of amino acids using a variety of reagents such as phenylisothiocyanate and o-phthalaldehyde, which react with the amino groups of the proteins. There are some inherent problems in derivatization of proteins prior to electrophoresis. In contrast to amino acids, which have one or two reaction sites, proteins can have multiple reactive sites producing multiple derivatization (heterogeneous) products with varying mobilities. This results in broadening of a protein peak. The production of heterogeneous derivatives can be minimized, to some extent, by using either mild or drastic derivatization conditions. In the former condition, only the most reactive sites will be derivatized, while in the latter condition all possible reactive sites will be labeled. 49
Capillary Coating
Capillary walls are coated in several ways in order to reduce the non-specific adsorption of protein onto the capillary wall. The capillary is generally deactivated by silanization, and the negatively charged silanol is then modified by a variety of groups such as methyl cellulose, polyacrylamide, polyethylene glycol, etc.
Capillary walls can be coated temporarily during electrophoresis by several buffer additives. High salts such as sulfates and phosphates of about 0.25 M compete with protein for adsorption, resulting in an improved separation. The only problem associated with high ionic strength buffer is the generation of Joule heat, which needs to be dissipated efficiently. Some zwitterionic salts such as betaine, sacrosine, and triglycine are shown to be advantageous up to 1 to 2 M without contributing significant change of conductivity. No single method is suitable for the separation of all types of proteins. Thus, the type of coating changes according to the nature of protein to be separated. For example, for the separation of hydrophobic proteins, non-ionic surfactants such as Tween 20 or Brij 35 are used to reduce the hydrophobicity of the coated capillary column. Similarly for the improved separation of the cationic proteins, the negative charge of the capillary wall is reversed by cationic surfactants such as CTAB.