Polypeptide subunits and their three-dimensional structure can be maintained in proteins by the presence of disulfide bonds. A goal of SDS PAGE analysis under reducing conditions is to disrupt this structure by reducing disulfide bonds. Complete denaturation and dissociation of proteins by treatment with 2-mercaptoethanol or dithiothreitol (DTT) will result in the unfolding of the polypeptide backbone and subsequent complexation with SDS. Under these conditions, the molecular weight of the polypeptide subunits can be calculated by linear regression in the presence of suitable molecular weight standards.

For some analyses, complete dissociation of protein to peptide subunits is not desirable. In the absence of treatment with reducing agents, disulfide covalent bonds remain intact, preserving the oligomeric form of the protein. Oligomeric SDS–protein complexes migrate more slowly than their SDS–polypeptide subunits. In addition, nonreduced proteins may not be completely saturated with SDS and hence may not bind the detergent in a constant weight ratio. This makes molecular weight determinations of these molecules less straightforward than analyses of fully denatured polypeptides, because, for valid comparisons, it is necessary that both standards and unknown proteins be in similar configurations. However, the staining of a single band in such a gel is a criterion of purity.

The most popular electrophoretic method for the characterization of a complex mixture of proteins involves the use of a discontinuous buffer system consisting of two contiguous, but distinct, gels: a resolving or separating (lower) gel and a stacking (upper) gel. The two gels are cast with different porosities, pHs, and ionic strengths. In addition, different mobile ions are used in the gel and electrode buffers. The buffer discontinuity concentrates large volumes of sample in the stacking gel, resulting in improved resolution. When power is applied, a voltage drop develops across the sample solution that drives the proteins into the stacking gel. Glycinate ions from the electrode buffer follow the proteins into the stacking gel. A moving boundary region is rapidly formed with the highly mobile chloride ions in the front and the relatively slow glycinate ions in the rear. A localized high-voltage gradient forms between the leading and trailing ion fronts, causing the SDS–protein complexes to form into a thin zone (stack) and migrate between the chloride and glycinate phases. Within a broad limit, regardless of the height of the applied sample, all SDS proteins condense into a very narrow region and enter the resolving gel as a well-defined, thin zone of high protein density. The large-pore stacking gel does not retard the migration of most proteins and serves mainly as an anticonvective medium. At the interface between the stacking and resolving gels, the proteins experience a sharp retardation due to the restrictive pore size of the resolving gel. Once in the resolving gel, proteins continue to be slowed by the sieving of the matrix. The glycinate ions overtake the proteins, which then move in a space of uniform pH formed by tris(hydroxymethyl)aminomethane (Tris) and glycine. Molecular sieving causes the SDS–polypeptide complexes to separate on the basis of their molecular weights.

In a discontinuous buffer SDS–polyacrylamide gel, it is important to pour the resolving gel, let the gel set, and then pour the stacking gel, because the composition of acrylamide–bisacrylamide, buffer, and pH are different.

30% Acrylamide–Bisacrylamide Solution— Prepare a solution containing 290 g of acrylamide and 10 g of methylene bisacrylamide per L of warm water, and filter. [note—Acrylamide and methylene bisacrylamide are slowly converted during storage to acrylic acid and bisacrylic acid, respectively. This deamidation reaction is catalyzed by light and alkali. The pH of the solution must be 7.0 or lower. Store the solution in dark bottles at room temperature. Fresh solutions are prepared every month.]

Ammonium Persulfate Solution— Prepare a small quantity of solution having a concentration of 100 g of ammonium persulfate per L, and store at 4. [note—Ammonium persulfate provides the free radicals that drive polymerization of acrylamide and bisacrylamide. Ammonium persulfate decomposes slowly; therefore, prepare fresh solutions weekly.]

TEMED— Use an electrophoresis-grade reagent. [note—TEMED accelerates the polymerization of acrylamide and bisacrylamide by catalyzing the formation of free radicals from ammonium persulfate. Because TEMED works only as a free base, polymerization is inhibited at low pH.]

SDS Solution— Use an electrophoresis-grade reagent. Prepare a solution having a concentration of about 100 g of SDS per L, and store at room temperature.

1.5 M Buffer Solution— Transfer about 90.8 g of Tris to a 500-mL flask, dissolve in 400 mL of water, adjust with hydrochloric acid to a pH of 8.8, dilute with water to volume, and mix.

1 M Buffer Solution— Transfer about 60.6 g of Tris to a 500-mL flask, add 400 mL of water, adjust with hydrochloric acid to a pH of 6.8, dilute with water to volume, and mix.

	Component Volume (mL) per Gel Mold Volume Below
Solution Component	5 mL	l0 mL	15 mL	20 mL	25 mL	30 mL	40 mL	50 mL
6% Acrylamide
Water	2.6	5.3	7.9	10.6	13.2	15.9	21.2	26.5
30% Acrylamide–Bisacrylamide Solution	1.0	2.0	3.0	4.0	5.0	6.0	8.0	10.0
1.5 M Buffer Solution	1.3	2.5	3.8	5.0	6.3	7.5	10.0	12.5
SDS Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
Ammonium Persulfate Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
TEMED	0.004	0.008	0.012	0.016	0.02	0.024	0.032	0.04
8% Acrylamide
Water	2.3	4.6	6.9	9.3	11.5	13.9	18.5	23.2
30% Acrylamide–Bisacrylamide Solution	1.3	2.7	4.0	5.3	6.7	8.0	10.7	13.3
1.5 M Buffer Solution	1.3	2.5	3.8	5.0	6.3	7.5	10.0	12.5
SDS Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
Ammonium Persulfate Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
TEMED	0.003	0.006	0.009	0.012	0.015	0.018	0.024	0.03
10% Acrylamide
Water	1.9	4.0	5.9	7.9	9.9	11.9	15.9	19.8
30% Acrylamide–Bisacrylamide Solution	1.7	3.3	5.0	6.7	8.3	10.0	13.3	16.7
1.5 M Buffer Solution	1.3	2.5	3.8	5.0	6.3	7.5	10.0	12.5
SDS Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
Ammonium Persulfate Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
TEMED	0.002	0.004	0.006	0.008	0.01	0.012	0.016	0.02
12% Acrylamide
Water	1.6	3.3	4.9	6.6	8.2	9.9	13.2	16.5
30% Acrylamide–Bisacrylamide Solution	2.0	4.0	6.0	8.0	10.0	12.0	16.0	20.0
1.5 M Buffer Solution	1.3	2.5	3.8	5.0	6.3	7.5	10.0	12.5
SDS Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
Ammonium Persulfate Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
TEMED	0.002	0.004	0.006	0.008	0.01	0.012	0.016	0.02
14% Acrylamide
Water	1.4	2.7	3.9	5.3	6.6	8.0	10.6	13.8
30% Acrylamide–Bisacrylamide Solution	2.3	4.6	7.0	9.3	11.6	13.9	18.6	23.2
1.5 M Buffer Solution	1.2	2.5	3.6	5.0	6.3	7.5	10.0	12.5
SDS Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
Ammonium Persulfate Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
TEMED	0.002	0.004	0.006	0.008	0.01	0.012	0.016	0.02
15% Acrylamide
Water	1.1	2.3	3.4	4.6	5.7	6.9	9.2	11.5
30% Acrylamide–Bisacrylamide Solution	2.5	5.0	7.5	10.0	12.5	15.0	20.0	25.0
1.5 M Buffer Solution	1.3	2.5	3.8	5.0	6.3	7.5	10.0	12.5
SDS Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
Ammonium Persulfate Solution	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
TEMED	0.002	0.004	0.006	0.008	0.01	0.012	0.016	0.02

	Component Volume (mL) per Gel Mold Volume Below
Solution Component	1 mL	2 mL	3 mL	4 mL	5 mL	6 mL	8 mL	10 mL
Water	0.68	1.4	2.1	2.7	3.4	4.1	5.5	6.8
30% Acrylamide–Bisacrylamide Solution	0.17	0.33	0.5	0.67	0.83	1.0	1.3	1.7
1.0 M Buffer Solution	0.13	0.25	0.38	0.5	0.63	0.75	1.0	1.25
SDS Solution	0.01	0.02	0.03	0.04	0.05	0.06	0.08	0.1
Ammonium Persulfate Solution	0.01	0.02	0.03	0.04	0.05	0.06	0.08	0.1
TEMED	0.001	0.002	0.003	0.004	0.005	0.006	0.008	0.01

Coomassie staining is the most common protein staining method, with a detection level on the order of 1 to 10 µg of protein per band. Silver staining is the most sensitive method for staining proteins in gels, because a band containing 10 to 100 ng can be detected; but the method is more cumbersome and less rugged. All of the steps in gel staining are performed at room temperature with gentle agitation (e.g., on a rocking platform shaker or equivalent). Gloves must be worn when staining the gels to prevent fingerprint residue staining.

Coomassie Staining Solution— Prepare a solution of Coomassie brilliant blue R-250 having a concentration of 1.25 g per L in a mixture of water, methanol, and glacial acetic acid (5:4:1). Filter, and store at room temperature.

Destaining Solution— Prepare a mixture of water, methanol, and glacial acetic acid (5:4:1).

Fixing Solution 1— Prepare a mixture of water, methanol, and trichloroacetic acid (5:4:1).

Fixing Solution 2— Transfer 250 mL of methanol to a 500-mL volumetric flask, add 0.27 mL of formaldehyde, dilute with water to volume, and mix.

Silver Nitrate Reagent— To a mixture of 40 mL of 1 M sodium hydroxide and 3 mL of ammonium hydroxide, add, dropwise and with stirring, 8 mL of a 200 g per L solution of silver nitrate; dilute with water to 200 mL, and mix.

Developing Solution— Transfer 2.5 mL of a citric acid solution (2 in 100) and 0.27 mL of formaldehyde to a 500.0-mL volumetric flask, dilute with water to volume, and mix.

Stopping Solution— Prepare a 10% (v/v) solution of acetic acid.

For Coomassie staining, after the destaining step, incubate the gel in a glycerol solution (1 in 10) for at least 2 hours. For silver staining, add to the final rinsing step a 5-minute incubation in a glycerol solution (1 in 50).

Immerse two sheets of porous cellophane in water, and incubate for 5 to 10 minutes. Place one of the sheets on a drying frame. Carefully lift the gel, and place it on the cellophane sheet. Remove any trapped air bubbles, and pour a few mL of water around the edges of the gel. Place the second sheet on top, and remove any trapped air bubbles. Complete the assembly of the drying frame. Place in a drying oven, leave at room temperature until dry, or use a commercial gel dryer.

Molecular weights of proteins are determined by comparison of their mobilities with those of several marker proteins of known molecular weight. Mixtures of proteins with precisely known molecular weights blended for uniform staining are available for calibrating gels. They are available in various molecular weight ranges. Concentrated stock solutions of proteins of known molecular weight are diluted in a sample buffer and loaded on the same gel as the protein sample to be tested.

Immediately after the gel has been run, the position of the bromophenol blue tracking dye is marked to identify the leading edge of the electrophoretic ion front. This can be done by cutting notches in the edges of the gel or by inserting a needle soaked in India ink into the gel at the dye front. After staining, measure the migration distances of each protein band (markers and unknowns) from the top of the Resolving Gel. Divide the migration distance of each protein by the distance traveled by the tracking dye. The normalized migration distances so obtained are called the relative mobilities of the proteins (relative to the dye front) and conventionally denoted as RF . Construct a (semilogarithmic) plot of the logarithm of the molecular weights (MR) of the protein standards as functions of the RF values. [note—The graphs are slightly sigmoid.] From the graph so obtained, estimate the unknown molecular weights by linear regression analysis or interpolation, as long as unknown samples are positioned along the linear part of the graph.

If the proteins of the molecular weight marker are not distributed along 80% of the length of the gel and over the required separation range (i.e., the range covering the product and its dimer or the products and its related impurities), and the separation obtained for the relevant protein bands does not show a linear relationship between the logarithm of the molecular weight and the RF , the test is not valid.

Where the impurity limit is specified in the individual monograph, a reference solution corresponding to that level of impurity is prepared by diluting the test solution. For example, where the limit is 5.0%, a reference solution is a 1 in 20 dilution of the test solution. No impurity—any band other than the main band—in the electrophoretogram obtained from the test solution is more intense than the main band obtained with the reference solution.

Under validated conditions and when using the Coomassie staining procedure, impurities may be quantified by normalization to the main band, using an integrating densitometer. In this case, the responses must be validated for linearity.