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3.7 ELECTROPHORESIS

3.7 ELECTROPHORESIS

         Electrophoresis is a physical method of analysis permitting the separation of compounds that are capable of acquiring an electrical charge in a conducting electrolyte. In this medium the ionized particles move more or less rapidly under the influence of an electrical field.

         The electrophoretic mobility is the rate of migration of the substance measured in cm/s under the influence of a potential gradient of 1 V/cm, and is expressed in cm2 V–1–1.

         The measurement of electrophoretic mobility is significant only where experimental conditions have been precisely defined. This mobility depends on the characteristics of the substance, its nature, size, form, and electrical charge. It also depends on the composition of the conducting liquid, its nature, concentration, pH, the presence of additional solvents, and viscosity. The direction of migration depends on the sign of the electrical charge of the particle as it moves towards the electrode of opposite sign.

         According to the methods used, the eletrophoretic mobility is either measured directly or compared with that of a reference substance.

Moving Boundary (Free-flow) Electrophoresis

         This technique, used exclusively for the determination of the mobility, is particularly suitable for substances of high molecular weight with poor diffusion properties.

         The boundaries are usually measured both before and after the application of an electrical field by a physical method, such as refractometry or conductometry. The concentration of the substance in the conducting liquid, the characteristics of the latter and the details of the procedure, including quantitative evaluation of the fractions, are specified in the monographs.

Zone Electrophoresis (Electrophoresis Using a Supporting Medium)

         This method uses only small sample sizes. The nature of the supporting medium (for example, paper, cellulose acetate, starch gel, agar gel, polyacrylamide gel, mixed gel) introduces additional factors influencing the mobility. The rate of migration depends on the mobility of the particles and also on the electroendosmotic current (in the case of carriers with polar properties), on the currents due to evaporation (caused by heat generated through the Joule effect), and on the gradient of the electrical field.

         In practice, the mobility of the electrophoretic zones and their signs are ignored; the zones are located by experience or by comparison with those given by a reference substance treated in the same way.

         After separation of the constituents, the position of colourless substances may be determined by treating the electrophoretogram with a reagent that will convert them to coloured or fluorescent derivatives. For quantitative purposes, the spot (zone) may be carefully separated, the substance eluted with a suitable solvent and then determined by a sufficiently sensitive method, such as spectrophotometric measurement, either directly or after a chemical reaction. In another quantitative procedure after conversion to a coloured derivative, the zone intensity can be measured with the aid of a scanning densitometer.

Apparatus

         The apparatus for electrophoresis consists essentially of the following components.

         (1) An appropriate power source supplying a constant, direct current and provided with means for indicating and controlling either the output voltage or the current consumption as appropriate; additional circuitry may be incorporated to stabilize the output.

          (2) An electrophoretic assembly with an appropriate support-carrying device.

         For electrophoresis using paper, agar gel, agarose gel, agarose-starch gel, or cellulose acetate as the electrophoretic support, the assembly consists of a tank with a close-fitting lid made of glass or other suitable material. The tank should be fitted with suitable safety devices to ensure that electrical supply is disconnected when the lid is removed. Two double troughs provided with a central lengthwise partition are inserted in the tank, one at each end; alternatively the troughs may be integral parts of the tank. One platinum electrode is laid along the bottom of one compartment of each double trough and the electrodes are connected through insulated leads, sealed through the walls of the tank, to external insulated cables which are connected to the power source. The troughs are filled with sufficient of the specified eletrolyte solution to ensure that the electrodes are fully immersed. Contact between the inner and outer compartment of each double trough is made either by means of “bridges” of electrophoresis paper or by perforating the central partition with several holes or by any other suitable method.

         For agar-gel, agarose-gel and agarose-starch-gel electrophoresis the tank is designed to allow ventilation that will prevent the condensation of moisture or the drying of the layer of the solid medium. For polyacrylamide-gel electrophoresis the assembly consists of two buffer reservoirs made of poly (methylmethacrylate) or similar material, each fitted with a platinum or graphite electrode. The upper reservoir is mounted vertically above the lower and its height is adjustable. It has a number of rubber holders in its base situated equidistant from the electrode. The electrodes are connected by insulated cables to the power source so that the cathode is in the upper reservoir and the anode in the lower.

         (3) An electrophoretic support. For paper and cellulose acetate electrophoresis the electrophoretic support is in the form of strips held between the troughs on a uniform surface composed of inert plastic or glass contact points, spaced so as to minimize capillary diffusion of the electrolyte solution.

         For agar-gel, agarose-gel and agarose-starch-gel electrophoresis the electrophoretic support is in the form of a gel, 6 mm thick, spread on a glass which in the case of agar-gel electrophoresis is placed on a hollow metal plate through which a cooling liquid may be circulated and the upper surface of which is machined to allow intimate contact with the glass plate.

         The electrophoretic support is connected either directly (paper and cellulose acetate electrophoresis) or indirectly by means of wicks of suitable material (agargel, agarose-gel and agarose-starch-gel electrophoresis) to the compartment of each double trough that does not contain the electrode.

         For polyacrylamide-gel electrophoresis the electrophoretic support is in the form of a gel contained in clean glass tubes, 7.5 cm long and 0.5 cm in internal diameter.

         (4) A measuring device or means of detection.

Methods

         Different methods in zone electrophoresis are described below.

PAPER ELECTROPHORESIS

          Fill the troughs of the tank with the electrolyte solution specified in the monograph.

         Apply separately to points along the line of application (drawn about 13 cm from one end of the electrophoresis paper), 1 cm from the edge of the paper and not less than 2.5 cm apart, the volumes of solutions prepared as described in the monograph.

         Allow the spots to dry and then place the end of the paper nearer the line of application in the appropriate compartment of the anode trough and the other end in the appropriate compartment of the cathode trough. Wet the paper with the electrolyte solution by a suitable method (for example using a brush, starting from the ends of the paper and working towards the line of application). Do not wet the strip that includes the applied samples. Close the lid, allow the electrolyte solution to diffuse across the line of application, if necessary cover the apparatus so as to exclude light, connect the cables to the power source and switch on the current. Adjust the voltage to about 20 V per cm of paper between the troughs and allow electrophoresis to proceed for the time indicated or until the marker substances have moved to the specified distances. Switch off the current, remove the paper, dry, if necessary in the dark, in a current of air, and examine under ultraviolet light (254 nm).

CELLULOSE ACETATE ELECTROPHORESIS

         Use Method I unless otherwise directed.

         Method I Fill the trough of the apparatus with the electrolyte solution specified in the monograph. Immerse cellulose acetate foil of suitable dimensions for 5 minutes in the same solution and press the strips dry between filter paper. Apply separately to the foil at points 1 cm from the anode edge and 2.5 cm apart 1 μl of each of the solutions prescribed in the monograph. Adjust the voltage to that specified in the monograph and allow electrophoresis to proceed for the specified time. Press the strips dry and immerse in a solution prepared by dissolving 1 g of potassium hexacyanoferrate (III) in 50 ml of water and adding 2 ml of a saturated solution of iron(III) chloride. Wash with a 5 per cent v/v solution of phosphoric acid until the background is as pale as possible and finally wash with water. Examine the electrophoretogram.

         Method II Fill the troughs of the apparatus with mixed barbital buffer pH 8.6. Using 10 separate strips of cellulose acetate for each solution prescribed in the monograph, apply either 2.5 μl of solution as a 10-mm band or, if narrower strips are used, 0.25 μl of solution per mm of strip width. Apply a suitable electric field such that the most rapid band migrates at least 30 mm. Stain the strips with a 0.5 per cent w/v solution of naphthalene black 12B in a mixture of 90 volumes of methanol and 10 volumes of 5 M acetic acid for 5 minutes and then decolorize with a mixture of 90 volumes of methanol and 10 volumes of acetic acid so that the background is just free of colour. Wash the strips with a mixture of 81 volumes of methanol and 19 volumes of 5 M acetic acid until the background is as transparent as possible. Measure the absorbance of the bands at 600 nm in an instrument having a linear response over the range of at least 0 to 3 (Appendix 2.2). Calculate the result as the mean of the measurements of each of the ten strips.

AGAR-GEL ELECTROPHORESIS

         Method I Place a suitable metal or plastic frame on the glass plate, seal its inner edge to the plate with a small quantity of liquefied Medium A. Place the plate on a level surface and pour on sufficient liquefied Medium A previously inoculated with 1 per cent v/v of the Inoculum of Test Organism A to produce a layer 1.2 to 1.6 mm thick. Allow the medium to set, remove the frame and bore at least 32 holes, about 1 mm in diameter, in the medium in such a manner that the solutions may be applied either in the form of a Latin Square or in a Randomized Block Design and that they are adequately spaced to allow the separation of the components.

         Place 5-μl quantities of each of the four solutions specified in the monograph in the holes in accordance with the chosen design. Transfer the prepared plate to the electrophoresis apparatus and place in each trough the same volume of Liquid Medium A, ensuring that the plate is level. Using wicks composed of a double layer of absorbent lint and moistened with Liquid Medium A, connect the contents of the appropriate compartment of each trough with the solid medium on the prepared plate; for the latter, the wicks should extend 2 cm across the solid medium and be pressed gently into contact with it. Close the tank and apply a potential between the electrodes to produce a voltage of 15 to 20 V per cm of the length of the layer of Medium A preferably using a stabilized voltage source. During electrophoresis circulate water or another suitable cooling liquid through the metal plate to prevent the temperature from rising above 15º. In atmospheres of high humidity, condensation of moisture may occur on the surface of the layer of Medium A if the ventilation of the box and cooling are not adequately controlled.

         Allow electrophoresis to proceed until separation of the substance to be determined has been attained. Disconnect the electrodes, remove the glass plate, cover it, and incubate at 30º to 35º for 18 hours, taking precautions to ensure that the layer of medium does not become dry. Measure the diameter, at right angles to the direction of migration, of the zones of inhibition produced by the reference solutions and any corresponding zones produced by the substance being examined. Calculate the result as a percentage of the substance being examined.

         Method II Carry out Method I with the following modifications.

         Use Medium B, Liquid Medium B and Inoculum of Test Organism B in place of Medium A, Liquid Medium A and Inoculum of Test Organism A, respectively.

         Apply a potential between the electrodes to produce a voltage of 25 to 30 V per cm of the length of the layer of Medium B. Allow electrophoresis to proceed for 2 hours.

AGAROSE-GEL ELECTROPHORESIS

         Use a glass plate of suitable dimensions over the surface of which is deposited a firmly adhering layer of uniform thickness of a gel prepared from Agarose for Electrophoesis. Use Electrolyte Solution I to equilibrate the agarose. Apply separately to the gel, 2 to 3 μl of the solutions prepared as described in the monograph. Close the tank, connect the electrodes to the power supply and allow electrophoresis to proceed at a current of 1 to 2 mA per cm of gel width at a potential of 300 V for about 10 minutes. Disconnect the electrodes, remove the glass plate and stain the gel using a 0.1 per cent w/v solution of Toluidine Blue removing the excess by washing. Evaluate the gels as prescribed in the monograph.

AGAROSE-STARCH-GEL ELECTROPHORESIS

         Use a glass plate of suitable dimensions. Place a suitable metal or plastic frame on the glass plate, seal its inner edge to the plate with a small quantity of liquefied Medium C, place the plate on a level surface, and pour on sufficient liquefied Medium C to produce a layer about 1 mm thick. Allow the medium to set, remove the frame and bore two holes 1 mm in diameter in the surface of the medium. The holes should be 5 cm apart and 5 cm from one end of the plate.

         Place 5-μl portions of each of the solutions specified in the monograph in the holes, pour into each compartment of the tank a sufficient volume of Electrolyte Solution II, and place the plate, gel uppermost, in the tank, ensuring that the plate is level. Using wicks composed of a double layer of absorbent lint and moistened with Electrolyte Solution II, connect the contents of the appropriate compartment of each trough with the solid medium on the prepared plate so that the cathode is connected to the end of the plate at which the holes are located; the wicks should extend 2 cm across the solid medium and be pressed gently into contact with it. Close the tank and apply a potential between the electrodes to produce a voltage of 20 V per cm of the layer of medium, preferably using a stabilized voltage source. Allow electrophoresis to proceed for 30 minutes, disconnect the electrodes and remove the glass plate. Using the same procedure as described above, cover the gel with a layer of liquefied Medium Apreviously inoculated with 1 per cent v/v of Inoculum of Test Organism A to produce a layer 1.2 to 1.6 mm thick. Incubate at 30º to 35º for 18 hours and measure the zones of inhibition produced.

POLYACRYLAMIDE ROD GEL ELECTROPHORESIS

         In polyacrylamide rod gel electrophoresis, the stationary phase is a gel which is prepared from a mixture of acrylamide and N,N’-methylenebisacrylamide. Rod gels are prepared in tubes 7.5 cm long and 0.5 cm in internal diameter, one solution being applied to each rod.

         Apparatus The platinum electrode is recommended.

         Procedure The solutions should usually be degassed before polymerization and the gels used immediately after preparation. Prepare the gel mixture as prescribed and pour into suitable glass tubes, stoppered at the bottom, to an equal height in each tube and to about 1 cm from the top, taking care to ensure that no air bubbles are trapped in the tubes. Cover the gel mixture with a layer of water to exclude air and allow to set. Gel formation usually takes about 30 minutes and is complete when a sharp interface appears between the gel and the water layer. Remove the water layer. Fill the lower reservoir with the prescribed buffer solution and remove the stoppers from the tubes. Fit the tubes into the holders of the upper reservoir and adjust so that the bottom of the tubes are immersed in the buffer solution in the lower reservoir. Carefully fill the tubes with the prescribed buffer solution. Prepare the test and reference solutions containing the prescribed marker dye and make them dense by dissolving in them sucrose, for example. Apply the solutions to the surface of a gel using a different tube for each solution. Add the same buffer to the upper reservoir. Connect the electrodes to the power supply and allow electrophoresis to proceed at the prescribed temperature and using the prescribed constant voltage or current. Switch off the power supply when the marker dye has migrated almost into the lower reservoir. Immediately remove each tube from the apparatus and extrude the gel. Locate the position of the bands in the electrophoretogram as prescribed.

SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)

         Polyacrylamide gel electrophoresis is used for the qualitative characterization of proteins in biological preparations, for control of purity and quantitative determinations.

         Analytical gel electrophoresis is an appropriate method with which to identify and to assess the homogeneity of proteins in pharmaceutical preparations. The method is routinely used for the estimation of protein subunit molecular masses and for determining the subunit compositions of purified proteins. Ready-touse gels and reagents are widely available on the market and can be used instead of those described in this text, provided that they give equivalent results and that they meet the validity requirements given below under Validation of the test.

         Characteristics of polyacrylamide gels The sieving properties of polyacrylamide gels are established by the three-dimensional network of fibres and pores which is formed as the bifunctional bisacrylamide cross-links adjacent polyacrylamide chains. Polymerization is catalyzed by a free radical-generating system composed of ammonium persulfate and tetramethylethylenediamine. As the acrylamide concentration of a gel increases, its effective pore size decreases. The effective pore size of a gel is operationally defined by its sieving properties; that is, by the resistance it imparts to the migration of macromolecules. There are limits on the acrylamide concentrations that can be used. At high acrylamide concentrations, gels break much more easily and are difficult to handle. As the pore size of a gel decreases, the migration rate of a protein through the gel decreases. By adjusting the pore size of a gel, through manipulating the acrylamide concentration, the resolution of the method can be optimized for a given protein product. Thus, a given gel is physically characterized by its respective composition in acrylamide and bisacrylamide. In addition to the composition of the gel, the state of the protein is an important component to the electrophoretic mobility. In the case of proteins, the electrophoretic mobility is dependent on the pK value of the charged groups and the size of the molecule. It is influenced by the type, concentration and pH of the buffer, by the temperature and the field strength as well as by the nature of the support material.

         Denaturing polyacrylamide gel electrophoresis The method cited as an example is limited to the analysis of monomeric polypeptides with a mass range of 14,000 to 100,000 daltons1 . It is possible to extend this mass range by various techniques (e.g., gradient gels, particular buffer system) but those techniques are not discussed in this appendix.

         Denaturing polyacrylamide gel electrophoresis using sodium dodecyl sulfate (SDS-PAGE) is the most common mode of electrophoresis used in assessing the pharmaceutical quality of protein products and will be the focus of the example method. Typically, analytical electrophoresis of proteins is carried out in polyacrylamide gels under conditions that ensure dissociation of the proteins into their individual polypeptide subunits and that minimize aggregation. Most commonly, the strongly anionic detergent sodium dodecyl sulfate (SDS) is used in combination with heat to dissociate the proteins before they are loaded on the gel. The denatured polypeptides bind to SDS, become negatively charged and exhibit a consistent charge-to-mass ratio regardless of protein type. Because the amount of SDS bound is almost always proportional to the molecular mass of the polypeptide and is independent of its sequence, SDSpolypeptide complexes migrate through polyacrylamide gels with mobilities dependent on the size of the polypeptide.

         The electrophoretic mobilities of the resultant detergent-polypeptide complexes all assume the same functional relationship to their molecular masses. Migration of SDS complexes is towards the anode in a predictable manner, with low-molecular-mass complexes migrating faster than larger ones. The molecular mass of a protein can therefore be estimated from its relative mobility in calibrated SDS-PAGE and the occurrence of a single band in such a gel is a criterion of purity.

         Modifications to the polypeptide backbone, such as N- or O-linked glycosylation, however, have a significant impact on the apparent molecular mass of a protein since SDS does not bind to a carbohydrate moiety in a manner similar to a polypeptide. Thus, a consistent charge-to-mass ratio is not maintained. The apparent molecular mass of proteins having undergone posttranslational modifications is not a true reflection of the mass of the polypeptide chain.

         REDUCING CONDITIONS Polypeptide subunits and three-dimensional structure is often 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)


One dalton is equivalent to 1.65 × 10–24 g. 

will result in unfolding of the polypeptide backbone and subsequent complexation with SDS. In these conditions, the molecular mass of the polypeptide subunits can be calculated by linear regression in the presence of suitable molecular-mass standards.

         NON-REDUCING CONDITIONS For some analyses, complete dissociation of the protein into subunit peptides is not desirable. In the absence of treatment with reducing agents such as 2-mercaptoethanol or DTT, disulfide covalent bonds remain intact, preserving the oligomeric form of the protein. Oligomeric SDS-protein complexes migrate more slowly than their SDSpolypeptide subunits. In addition, non-reduced proteins may not be completely saturated with SDS and, hence, may not bind the detergent in a constant mass ratio. This makes molecular-mass determinations of these molecules by SDS-PAGE less straightforward than analyses of fully denatured polypeptides, since it is necessary that both standards and unknown proteins be in similar configurations for valid comparisons. However, the staining of a single band in such a gel is a criterion of purity.

         Characteristics of discontinuous buffer system gel electrophoresis The most popular electrophoretic method for the characterization of complex mixtures 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, pH, and ionic strengths. In addition, different mobile ions are used in the gel and electrode buffers. The buffer discontinuity acts to concentrate large volume samples in the stacking gel, resulting in improved resolution. When power is applied, a voltage drop develops across the sample solution which 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 broad limits, regardless of the height of the applied sample, all SDSproteins 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 of the stacking and resolving gels, the proteins experience a sharp increase in 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 the tris- (hydroxymethyl) aminomethane and glycine. Molecular sieving causes the SDS-polypeptide complexes to separate on the basis of their molecular masses.

Preparing vertical discontinuous buffer SDS polyacrylamide gels

         ASSEMBLING OF THE GEL MOULDING CASSETTE Clean the two glass plates (size: e.g., 10 cm × 8 cm), the polytetrafluoroethylene comb, the two spacers and the silicone rubber tubing (diameter: e.g., 0.6 mm × 35 cm) with mild detergent and rinse extensively with water. Dry all the items with a paper towel or tissue. Lubricate the spacers and the tubing with non-silicone grease. Apply the spacers along each of the two short sides of the glass plate 2 mm away from the edges and 2 mm away from the long side corresponding to the bottom of the gel. Begin to lay the tubing on the glass plate by using one spacer as a guide. Carefully twist the tubing at the bottom of the spacer and follow the long side of the glass plate. While holding the tubing with one finger along the long side twist again the tubing and lay it on the second short side of the glass plate, using the spacer as a guide. Place the second glass plate in perfect alignment and hold the mould together by hand pressure. Apply two clamps on each of the two short sides of the mould. Carefully apply four clamps on the longer side of the gel mould thus forming the bottom of the gel mould. Verify that the tubing is running along the edge of the glass plates and has not been extruded while placing the clamps. The gel mould is now ready for pouring the gel.

         PREPARATION OF THE GEL In a discontinuous buffer SDS polyacrylamide gel, it is recommendesd to pour the resolving gel, let the gel set, and then pour the stacking gel since the composition of the two gels in acrylamidebisacrylamide, buffer and pH are different.

         Preparation of the resolving gel In a conical flask, prepare the appropriate volume of solution containing the desired concentration of acrylamide for the resolving gel, using the values given in Table 1. Mix the components in the order shown. Where appropriate, before adding the ammonium persulfate solution and the teramethylethylenediamine (TEMED), filter the solution if necessary under vacuum through a cellulose acetate membrane (pore diameter: 0.45 μm); keep the solution under vacuum by swirling the filtration unit until no more bubbles are formed in the solution. Add appropriate amounts of ammonium persulfate solution and TEMED as indicated in Table 1, swirl and pour immediately into the gap between the two glass plates of the mould. Leave sufficient space for the stacking gel (the length of the teeth of the comb plus 1 cm). Using a tapered glass pipette, carefully overlay the solution with watersaturated 2-methyl-1-propanol. Leave the gel in a vertical position at room temperature to allow polymerization.

         Preparation of the stacking gel After polymerization is complete (about 30 minutes), pour off 2-methyl-1-propanol and wash the top of the gel several times with water to remove the 2-methyl-1-propanol overlay and any unpolymerized acrylamide. Drain as much fluid as possible from the top of the gel, and remove any remaining water with the edge of a paper towel. In a conical flask, prepare the appropriate volume of solution containing the desired concentration of acrylamide, using the values given in Table 2. Mix the components in the order shown. Where appropriate, before adding the ammonium persulfate solution and the TEMED, filter the solution if necessary under vacuum through a cellulose acetate membrane (pore diameter: 0.45 μm); keep the solution under vacuum by swirling the filtration unit until no more bubbles are formed in the solution.

         Add appropriate amounts of ammonium persulfate solution and TEMED as indicated in Table 2, swirl and pour immediately into the gap between the two glass plates of the mould directly onto the surface of the polymerized resolving gel. Immediately insert a clean polytetrafluoroethylene comb into the stacking gel solution, being careful to avoid trapping air bubbles. Add more stacking gel solution to fill the spaces of the comb completely. Leave the gel in a vertical position and allow to polymerize at room temperature.

         MOUNTING THE GEL IN THE ELECTROPHORESIS ELECTROPHORESIS APPARATUS AND ELECTROPHORETIC SEPARATION After polymerization is complete (about 30 minutes), remove the polytetrafluoroethylene comb carefully. Rinse the wells immediately with water or with the SDSPAGE running buffer to remove any unpolymerized acrylamide. If necessary, straighten the teeth of the stacking gel with a blunt hypodermic needle attached to a syringe. Remove the clamps on one short side,carefully pull out the tubing and replace the clamps. Proceed similarly on the other short side. Remove the tubing from the bottom part of the gel. Mount the gel in the electrophoresis apparatus. Add the electrophoresis buffers to the top and bottom reservoirs. Remove any bubbles that become trapped at the bottom of the gel between the glass plates. This is best done with a bent hypodermic needle attached to a syringe. Never prerun the gel before loading the samples, since this will destroy the discontinuity of the buffer systems. Before loading the sample, carefully rinse the slot with SDSPAGE running buffer. Prepare the test and reference solutions in the recommended sample buffer and treat as specified in the individual monograph. Apply the appropriate volume of each solution to the stacking gel wells. Start the electrophoresis using the conditions recommended by the manufacturer of the equipment. Manufacturers of SDS-PAGE equipment may provide gels of different surface area and thickness. Electrophoresis running time and current/voltage may need to vary as described by the manufacturer of the apparatus in order to achieve optimum separation. Check that the dye front is moving into the resolving gel. When the dye is reaching the bottom of the gel, stop the electrophoresis. Remove the gel assembly from the apparatus and separate the glass plates. Remove the spacers, cut off and discard the stacking gel and immediately proceed with staining.

         Detection of proteins in gels Coomassie staining is the most common protein staining method with a 

Table 1 Preparation of Resolving Gel

*a 100 g/l solution of sodium dodecyl sulfate.

**a 100 g/l solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solutions must be prepared weekly.

 tetramethylethylenediamine. 

Table 2 Preparation of Stacking Gel​

*a 100 g/l solution of sodium dodecyl sulfate.

**a 100 g/l solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solutions must be prepared weekly.  tetramethylethylenediamine.

detection level of the order of 1 to 10 μg of protein per band. Silver staining is the most sensitive method for staining proteins in gels and a band containing 10 to 100 ng can be detected.

         All of the steps in gel staining are done at room temperature with gentle shaking (e.g., on an orbital shaker platform) in any convenient container. Gloves must be worn when staining gels, since fingerprints will stain.

         COOMASSIE STAINING Immerse the gel in a large excess of Coomassie staining solution and allow to stand for at least 1 hour. Remove the staining solution.

         Destain the gel with a large excess of Destaining solution. Change the destaining solution several times, until the stained protein bands are clearly distinguishable on a clear background. The more thoroughly the gel is destained, the smaller is the amount of protein that can be detected by the method. Destaining can be speeded up by including a few grams of anion-exchange resin or a small sponge in the Destaining solution. (Note The acid-alcohol solutions used in this procedure do not completely fix proteins in the gel.) This can lead to losses of some low-molecular-mass proteins during the staining and destaining of thin gels. Permanent fixation is obtainable by allowing the gel to stand in a mixture of 1 volume of trichloroacetic acid, 4 volumes of methanol and 5 volumes of water for 1 hour before it is immersed in the Coomassie staining solution.

         SILVER STAINING Immerse the gel in a large excess of fixing solution and allow to stand for 1 hour. Remove the fixing solution, add fresh fixing solution and incubate either for at least 1 hour or overnight, if convenient. Discard the fixing solution and wash the gel in a large excess of water for 1 hour. Soak the gel for 15 minutes in a 1 per cent v/v solution of glutaraldehyde. Wash the gel twice for 15 minutes in a large excess of water. Soak the gel in fresh Silver staining solution for 15 minutes, in darkness. Wash the gel three times for 5 minutes in a large excess of water. Immerse the gel for about 1 minute in Developing solution until satisfactory staining has been obtained. Stop the development by incubation in the Blocking solution for 15 minutes. Rinse the gel with water.

         Drying of stained SDS polyacrylamide gels Depending on the staining method used, gels are treated in a slightly different way. For Coomassie staining, after the destaining step, allow the gel to stand in a 10 per cent w/v solution of glycerol for at least 2 hours (overnight incubation is possible). For silver staining, add to the final rinsing a step of 5 minutes in a 2 per cent w/v solution of glycerol. Immerse two sheets of porous cellulose film 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 cellulose film. Remove any trapped air bubbles and pour a few millilitres 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 an oven or leave at room temperature until dry.

         Molecular-mass determination Molecular masses 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 masses blended for uniform staining are available for calibrating gels. They are obtainable in various molecular mass ranges. Concentrated stock solutions of proteins of known molecular mass are diluted in the appropriate sample buffer and loaded on the same gel as the protein sample to be studied. 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 travelled 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 plot of the logarithm of the relative molecular masses (Mr) of the protein standards as a function of the Rf values. Note that the graphs are slightly sigmoid. Unknown molecular masses can be estimated by linear regression analysis or interpolation from the curves of log Mr against Rf as long as the values obtained for the unknown samples are positioned along the linear part of the graph.

         Validation of the test The test is not valid unless the proteins of the molecular mass marker are distributed along 80 per cent of the length of the gel and over the required separation range (e.g., the range covering the product and its dimer or the product and its related impurities) the separation obtained for the relevant protein bands shows a linear relationship between the logarithm of the molecular mass and the Rf. Additional validation requirements with respect to the solution under test may be specified in individual monographs.

          Quantification of impurities Where the impurity limit is specified in the individual monograph, a reference solution corresponding to that level of impurity should be prepared by diluting the test solution. For example, where the limit is 5 per cent, a reference solution would be a 1:20 dilution of the test solution. No impurity (any band other than the main band) in the electropherogram obtained with the test solution may be more intense than the main band obtained with the reference solution.

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

SAFETY PRECAUTIONS

         Voltages used in electrophoresis can readily deliver a lethal shock. The hazard is increased by the use of aqueous buffer solutions and the possibility of working in damp environments.

         The equipment, with the possible exception of the power supply, should be enclosed in either a grounded metal case or a case made of insulating material. The case should have an interlock that de-energizes the power supply when the case is opened, after which reactivation should be prevented until activation of a reset switch is carried out.

         High-voltage cables from the power supply to the apparatus should preferably be a type in which a braided metal shield completely encloses the insulated central conductor, and the shield should be grounded. The base of the apparatus should be grounded metal or contain a grounded metal rim which is constructed in such a way that any leakage of electrolyte will produce a short which will de-energize the power supply before the electrolyte can flow beyond the protective enclosure.

         If the power supply contains capacitors as part of a filter circuit, it should also contain a bleeder resistor to ensure discharge of the capacitors before the protective case is opened. A shorting bar that is activated by opening the case may be considered as an addedprecaution.

         Because of the potential hazard associated with electrophoresis, laboratory personnel should be completely familiar with electrophoresis equipment before using it.

REAGENTS

         Acrylamide solution Prepare a solution containing 290 g of acrylamide and 10 g of methylenebisacrylamide per litre of water and filter.

         Agarose for electrophoresis Use electrophoretic grade of commerce.

         Blocking solution A 10 per cent v/v solution of acetic acid.

         Coomassie staining solution A 0.125 per cent w/v solution of acid blue 83 in a mixture of 1 volume of glacial acetic acid, 4 volumes of methanol and 5 volumes of water.

         Destaining solution A mixture of 1 volume of glacial acetic acid, 4 volumes of methanol and 5 volumes of water.

         Developing solution

Formaldehyde Solution          0.30 ml

Citric Acid                             0.025 g

Water sufficient to produce    500.0 ml

         Electrolyte solution I Mix 50 ml of glacial acetic acid and 800 ml of water, adjust the pH to 3.0 with lithium hydroxide and dilute to 1000 ml with water.

         Electrolyte solution II Dissolve 1.015 g of dipotassium hydrogenphosphate and 340 mg of potassium dihydrogenphosphate in sufficient water to produce 1000 ml.

         Electrophoresis paper Suitable filter paper (Whatman 3 MM or equivalent is suitable) that has been washed chromatographically for 16 hours with a mixture of 2 volumes of acetone and 1 volume of water. After drying, cut the paper into strips of appropriate size.

         Fixing solution To 250 ml of methanol and 0.27 ml of formaldehyde solution and dilute with sufficient water to produce 500 ml.

         Inoculum of test organism A Grow Bacillus subtilis (ATCC 11774, NCTC 8236) at a temperature of 37º to 39º for 7 days on the surface of Medium A to which has been added 0.001 per cent w/v of manganese(II) sulfate. Using sterile water, wash off the growth, which consists mainly of spores, and dilute to give a suitable suspension; the degree of dilution should be determined experimentally. A suitable suspension usually contains between 107 and 108 spores per ml. The suspension may be stored for long periods at a temperature not exceeding 4º.

         Inoculum of test organism B Prepare as for Inoculum of test organism A but using Medium B in place of Medium A.

         Medium A

Dried peptone 6.0 g

Pancreatic digest of casein 4.0 g

Beef extract 1.5 g

Yeast extract 3.0 g

Dextrose monohydrate 1.0 g   

Agar 15.0 g

Water sufficient to produce 1000 ml.

         Sterilize under pressure at 121º for 15 minutes. Immediately before use adjust to pH 6.5 by the addition of 0.1 M hydrochloric acid

         Liquid medium A Prepare as described for Medium A using the same ingredients but omitting the agar.

         Medium B

Dried peptone 3.0 g

Pancreatic digest of casein 2.0 g

Beef extract 0.75 g

Yeast extract 1.5 g

Agar 10.0 g

Water sufficient to produce 1000 ml

         Sterilize under pressure at 121º for 15 minutes. Immediately before use adjust to pH 6.5 by the addition of 0.1 M hydrochloric acid.

         Liquid medium B Prepare as described for Medium B, using the same ingredients but omitting the agar.

         Medium C

Agarose for electrophoresis 10.0 g

Hydrolyzed starch 10.0 g

Electrolyte solution II sufficient to produce 1000 ml

         Add the Agarose for Electrophoresis and Hydrolyzed Starch to the electrolyte solution and heat in saturated steam at 121º until they are dissolved. Maintain the molten gel at a temperature of 50º until required for use.

         Pretreatment solution Mix together 250 ml of methanol, 250 ml of water and 0.1 ml of formaldehyde solution.

         Sample application buffer

Tris(hydroxymethyl)methylamine 1.5 g

1 M Hydrochloric acid 12.0 ml

Urea 96.0 g

Water sufficient to produce 200.0 ml

Sample buffer

Tris(hydroxymethyl)methylamine 0.76 g

Glycerol 5.0 ml

Sodium dodecyl sulfate 1.0 g

Bromophenol blue 0.1 g

2-Mercaptoethanol 1.0 ml

Water 25.0 ml

         Mix together and adjust the pH to 6.8 with 6 M hydrochloric acid and dilute to 50.0 ml with water.

         Silver staining solution Mix 40 ml of 1 M sodium hydroxide with 3 ml of strong ammonia solution, add 8 ml of a 20 per cent w/v solution of silver nitrate, and dilute with sufficient water to produce 200 ml.

         Solution A

Tris(hydroxymethyl)methylamine 36.6 g

N,N,N’,N’-Tetramethylethylenediamine 0.23 ml

1 M Hydrochloric acid 48.0 ml      

Water sufficient to produce 100.0 ml

         Solution B

Acrylamide 30.0 g

N,N’-Methylenebisacrylamide 0.735 g

Water sufficient to produce 100.0 ml

         Solution C Dissolve 6.0 g of tris(hydroxymethyl)- methylamine in 70 ml of water. Adjust the pH to 8.8 with 6 M hydrochloric acid and dilute with sufficient water to produce 100.0 ml.

         Solution D Dissolve 6.0 g of tris(hydroxymethyl)- methylamine in 70 ml of water. Adjust the pH to 6.8 with 6 M hydrochloric acid and dilute with sufficient water to produce 100.0 ml.

         Starch, hydrolyzed Use electrophoretic grade of commerce.

         1.0 M Tris (pH 6.8) Dissolve 60.6 g of tris (hydroxymethyl)aminomethane in 400 ml of water, adjust the pH with hydrochloric acid and dilute to 500 ml with water.

         1.5 M Tris (pH 8.8) Dissolve 90.8 g of tris (hydroxymethyl)methylamine in 400 ml of water, adjust the pH with hydrochloric acid and dilute to 500 ml with water.

         Toluidine blue (Toluidine blue O) C15H16ClN3S = 305.83

         DESCRIPTION Dark green powder.

         SOLUBILITY Soluble in water; slightly soluble in ethanol.

APPENDICES • 3.7 ELECTROPHORESIS
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หมายเหตุ / Note : TP II 2011 PAGE 399-408