One-way gel electrophoresis experiment
One-way gel electrophoresis experiment
The S D S -- P A G E methods presented in this book will fulfill the needs of most applications in this field.
Authors: Burgess et al, Translator: Chen Wei, This experiment is from the "Guide to Protein Purification".
Operation method
One-way gel electrophoresis experiment Move I. Steps 1. Reserve solution (1) Acrylamide Concentrate ( 30 % T, 2. 7 % C: Dissolve 29. 2 g of acrylamide and 0. 8 g of bisacrylamide in 70 m L of deionized water. When the acrylamide is completely dissolved, add water to volume to IOO mL. The solution was vacuum filtered using a 0.45 um filter membrane. Store the acrylamide stock solution in a dark-colored bottle at 4°C for a period of time not exceeding (2) I.5 m o l / L Tris - H C U p H 8. 8, Concentrated Separation Gel Buffer: Dissolve 18.2 g of Tris base in about 80 m L of water, adjust the p H to 8.8 with H C U, and add water to volume to 100 m L. Store at 4°C. Store at 4°C. (3) 0.5 mOl/L Tris-HCU pH 6.8, Concentrated Layer Gel Buffer: Dissolve 6.2 g of Tris base in approximately 80 mL of water, adjust pH to 6.8, and add water to 100 mL. Store at 4°C. (4) 10% (m/V) Sodium Dodecyl Sulfate (SDS): Dissolve 10 g of SDS in approximately 60 mL of water, then add water to 100 mL. (5) Sample stock buffer [0.6 m o l / L]. 0.06 m o l / L Tris-H C l (p H 6. 8), 2 % SDS, 10 % glycerol, 0.025 % bromophenol blue]. Electrophoresis buffer: 0.025 moI/L Tris, 0.192 mol/L Glycine, 0.1 % (m/V) SDS, pH 8.3 (0.3 g Tris, 1.4 g Glycine, 10 % SDS per 100 mL of electrophoresis buffer). There is no need to adjust the pH of the electrophoresis buffer; just dissolve the reagents together and make sure the pH is around 8.3 (±0.2). The electrophoresis buffer can be prepared as a 5 X concentrate containing 15 g Tris base, 72 g Glycine and 5 g SDS per liter. The 5 X electrophoresis buffer concentrate must be stored in a glass container. Prior to use, dilute with 4 times the volume of water. Thoroughly clean the glass plates, gaskets, tooth combs, and the upper buffer tank of the gel unit with a decontaminating agent and rinse well with water. Wear gloves when assembling the device. First fill with separation gel, then cover with build-up gel. (1) Assemble the pouring device and determine the amount of gel according to the manufacturer's instructions or by calculation. Use 1 to 2 cm of laminating gel on top of the separation gel. Determine the height of the poured separation gel by inserting a well-shaped tooth comb between the two glass plates and making the outer glass plate 1 to 2 cm lower than the teeth of the tooth comb. (2) Mix all the reagents in Table 29.1 except APS and TEMED to prepare a suitable solution of the separating gel monomer; a disposable plastic beaker is a convenient mixing vessel. Two gel formulations are provided in Table 29.1 to meet the different molecular mass requirements typically encountered. To prepare acrylamide gels at other concentrations (Andrews, 1986; Blackshear, this series; Hames, 1981), simply adjust the amounts of 30% monomer stock solution and water used in the formulation. (3) Gently mix APS and TEMED (Table 29.1) into the degassed monomer solution. Using a pipette and a pipette bulb, add the monomer solution between the gel glass plates up to the mark at the boundary of the separating gel. Immediately cover the monomer solution with an aqueous saturated solution of sec-butanol or tert-pentanol to exclude air and to prevent antipolymerization due to the presence of air on the surface of the monomer mixture. Allow the gel to polymerize for 45 min to I h. After about 15 min the underside of the overlayer is covered. After about 15 min, a distinct interface will begin to appear underneath the cover layer, proving that polymerization is occurring. In essence, polymerization takes about 90 min to complete, but 1 h to complete. (4) Prepare 10 m L of cumulus monomer solution (4% t, 2. 7 % C) by mixing the following components. Water 6.1 m L 0.5 m o l / L Tris-H C l ,p H 6. 8 2.5 m L Acrylamide stock solution ( 30 % T ) 1.3 m L 10 % S D S 0.1 m L Vacuum place at least 15 m i n to remove air bubbles from the monomer solution. (5) Thoroughly rinse the top of the separation gel with water and place filter paper over this to absorb the moisture. Place well-shaped toothed combs between the gel glass plates so that they are tilted at a slight angle to allow air bubbles to escape smoothly. (6) Add 50 A 1 0 % A P S and 10 A T E M E D per 10 m L of degassing monomer solution and pour the cumulus gel solution over the top of the separation gel. Make the tooth combs line up in their proper positions, taking care not to trap air bubbles in the lower ends of the teeth . Visible polymerization of the cumulus gel should occur within about 10 m i n . Since the combs have already removed oxygen from the surface of the perforations, no further covering is required. Allow 30 to 45 m i n for gel polymerization. The unused monomer is polymerized in a beaker and discarded. In some cases, it may be necessary to leave the gel overnight before using it. In this case, it is best to pour the cumulus gel on the day the gel is used in order to maintain the discontinuity of the interfacial ions between the two gels. When storing, the top of the separating gel should be thoroughly washed and covered with separating gel buffer (0.375 m o l / L Tris-H C K O .1 % S D S ,p H 8. 8) to prevent dehydration and ion depletion of the gel. Also, the gel along with the top of the splint needs to be wrapped in plastic wrap during storage. Sample preparation is generally not necessary because commonly used biochemical buffers are usually resistant to S D ^ P A G E . Band distortions, such as contraction or expansion of bands, may be caused by excessive salt content in the sample. Desalting the sample can remedy these distortions. (1) Prepare the required amount of SDS-reducing buffer for the samples to be tested by adding 50 ul of 2-mercaptoethanol (final concentration of 2-mercaptoethanol is 5 %) per 0.95 mL of sample stock buffer. (2) Dilute the sample with at least 4 times the volume of full SDS-Reducing Buffer (although for some samples, 2 times the volume is sufficient). The sample volume depends on the width of the wells and the thickness of the gel, with 20-50 uL for regular gels and 5-30 uL for minigels. For easy visualization of the bands during staining, approximately I ug (R-250 staining with koji) or 0.1ug (silver staining) of protein should be added to each band (see below). (3) Suspend the tube containing the sample in a hot water bath and heat the diluted sample at 95°C for 4 m i n . Do not store the prepared sample. Assemble the electrophoresis apparatus, add electrophoresis buffer to the upper and lower cells, and remove the tooth combs from the laminar gel. Using a microsampler or micropipette, load the prepared samples into the wells of the laminar gel so that they form a layer at the bottom of the electrophoresis buffer. The glycerol in the sample provides the necessary density conditions for the sample to sink to the bottom of the wells, and the bromophenol blue tracer dye makes the sample loading process visible. Finally , the wires are connected to the electrophoresis unit and the power is turned on. In S D S -P A G E , the lower electrode is the anode and the upper electrode is the cathode. During electrophoresis, electrical energy is converted into thermal energy and causes deformation and spreading of the strips. Normally, electrophoresis is carried out at a voltage that enables the electrophoresis to run as fast as possible, within the limits of the heat dissipation capacity of the electrophoresis bath. In other words, the faster the electrophoresis rate, the better, without compromising the desired separation and without causing band distortion. Many of the available power supplies can control the amount of power, and the choice is mostly a matter of preference. In general, a constant current will result in shorter electrophoresis times but more heat production than a constant voltage (Alien et al., 1984). At the beginning of electrophoresis, the resistance of the gel increases as chloride ions migrate out. Therefore, the voltage rises when using constant current electrophoresis and the current falls when using constant voltage electrophoresis. A small version of the electrophoresis casket with a thin glass plate is more efficient than a conventional-sized tank in dissipating the heat generated by the high current at the beginning of electrophoresis. Therefore, we recommend that gels be electrophoresed in a conventional electrophoresis apparatus with a constant current (16 to 24 m A /m m gel thickness) and in a mini electrophoresis apparatus with a constant voltage (200 to 30 V /c m gel length). Recirculating coolants can also be used to reduce electrophoresis time by increasing voltage and current. Electrophoresis should begin immediately after sampling and usually continues until the bromophenol blue tracer dye reaches the bottom of the gel. The SDS-PAGE method of Lameuli (Alien et al., 1984; Arndrews, 1986; Blackshear, this series; Hames, 1981; Lameuli, 1970) is an improved version of the earlier method applied by Ornstein (1964) and Davis (1964) for the isolation of natural serum proteins. The Ornstein-Davis system The Ornstein-Davis system (Ornstein, 1964; Jovin, 1973) uses different (discontinuous) buffers for the laminating and separating gels, which are required for the proper functioning of the system. However, the S D S adders made important modifications to the basic principles of the O m s t e i n -Davis technique because of the qualities of this decontaminant that dominate the entire system (Allen et al., C h r a m b a c h , 1 9 8 5 ; Wyckoff et al., 1 9 7 7 ) . The necessary components of the L a e m m l i S D S- P A G E system include T r i s - H C l gel buffer, Tris-glycine-S D S electrophoresis buffer, and S D S reduction sample buffer. With the addition of S D S to the system, it is not practically necessary to take a different p H and ionic strength for the preparation of the cumulus gel than for the separator gel. Similar resolution can be observed in the preparation of the gel, regardless of whether the conditions are as described above or whether the separator buffer (0.375 mOl/L Tris-HCU pH 8.8) is used. This is because the motion of the S D S ■ peptide complexes in this range is insensitive to p H (Allen et al., 1984). When many gels are prepared at once to be stored for later use, it is convenient to use the same buffer for both cumulus and separator gels. In addition, the complete incorporation of S D S has a significant effect on resolution (W y c k o f f et al., 1977). In a microelectrophoresis setup, the addition of more than 200 Fg of SDS to 30-50 samples results in broadening and spreading of protein bands. For dilute, large-volume samples, it is beneficial to limit the total amount of SDS in the system by reducing the final concentration of SDS in the processed samples to 0.5 % and by not adding SDS during gel preparation. Since the mobility of SDS is greater than that of protein, the amount of SDS in the electrophoresis buffer quickly exceeds that of protein during electrophoresis. Therefore, in order to maintain SDS at a level sufficiently saturated with protein, the gel must be able to receive a constant replenishment of SDS from the electrophoresis buffer (C h r a m b ac h , 1 9 85). L a e m m l i S D S - P A G E system (Allen et al. , 1984; A n d r e w s , 1986; Blackshear, this series; H a m eS, 1981; L aem m l i , 1970) in which fully denatured and decomposed proteins are not always the most suitable. For some analyses, one's interest may be in determining the molecular mass of individual intact, oligomeric forms of proteins. In other experiments, interest has focused on the biological activity of proteins in their natural, nondenatured state. With the choice of two denaturants, 2-mercaptoethanol and SDS, conditions can be adjusted as needed to isolate fully denatured, partially denatured, or naturally occurring proteins. Removal of 2-mercaptoethanol from the sample buffer maintains the covalent linkage between protein subunits. In the absence of the reducing agent, intra- and inter-chain disulfide bonds of the sample protein remain intact. The electrophoretic mobility of the resulting SDS-protein complexes changes accordingly compared to those obtained under dissociative conditions. The mobility of oligomeric SDS proteins is lower than that of their fully denatured SDS-polypeptide fractions during electrophoresis. In addition, the electrophoretic behavior of single-chain polypeptides is also affected by the reducing agent. The intrachain disulfide bonds of single-chain proteins maintain their tight conformation in the presence of SDS. In order to isolate proteins without reductants, the SDS-PA G E procedure described above can be developed and there is no need to add 2-mercaptoethanol to the sample buffer. It should be noted that the oligo-SDS-methionine complexes migrate more slowly than their SD^ peptide subunits. Therefore, it may be necessary to use a lower concentration (% T) of gel than that used in the full denaturation method in order to move the oligomers an appropriate distance in the medium. In addition, unreduced proteins may not be fully saturated by the SDS, and therefore such proteins may not bind the detergent at a constant gravity ratio. This makes the determination of the molecular mass of such molecules by the SD^PAGE method less clear than that of fully denatured peptides, and therefore the molecular mass criteria for proteins must be structurally the same as those for unknown proteins in order to make a valid comparison. The classic ◦ rnstein-Davis PAGE method for the detection of natural proteins is based on the removal of SDS and 2-mercaptoethanol from the Lamemlie method (① avis, 1964; ○ rnstein, 1964). It is a high-resolution natural PAGE method designed to separate the full spectrum of serum proteins. Since this system is intended to separate a wide range of proteins, its resolution does not apply to the limited range of migration of certain proteins. Although there are a number of natural PAGE systems with high resolution that can be adapted to meet different needs (Allen et al., 1984; Andrews, 1986; Blackshear, this series; Charmabach, 1985; Hammes, 1981), the Ornstein-Davis method is sufficiently well adapted to the separation of most commonly encountered protein mixtures (e.g., the Ornstein-Davis method). The Ornstein-Davis method is adequate for the separation of most commonly encountered proteinaceous mixtures. The natural PAGE method is more difficult to determine the molecular mass than the SDS-PAGE method because a single natural system cannot distinguish between the effects of charge and conformational factors in the electrophoretic movement of proteins. The steps described here are a simpler modification of the natural PAGE method. Simply remove the 2-mercaptoethanol from the sample buffer and replace the 10% SDS in the gel, sample and buffer formulations with an equal volume of water. Except for sample treatment, follow the above steps unless otherwise specified. Samples should be prepared in a non-denaturing buffer [0.6 ml/L]. The sample should be diluted in a non-denaturing buffer solution [106 ml/LTris - HClPH 6.8), 10% glycerol, 0-025% bromoglycolic acid blue] following the guidelines for sample preparation for denaturing gel electroswimming, except that the sample should not be heated. We have provided three of the simplest and most reliable methods for the detection of protein in SDS-PAGE gels, which are adequate for most situations. Caulmers Brilliant Blue R-250 is the most commonly used protein stain and is recommended for routine testing. Silver staining is the most sensitive method for protein staining in gels and should be used when evaluating the purity of preparations by electroswimming (e.g. in antigen preparation). Copper staining is a rapid and sensitive staining method that has been developed recently. Discussions on other detection methods, including radioactive labeling and protein quantification in gels, can also be found in the literature by Dumbard (1987), Andrews (1986), Alien et al. (1984), Hames (1981), and Merril (this book). After electrophoresis, remove the gel apparatus and separate the glass plates. The gel may stick to one of the glass plates. Remove the spacer strips, excise and discard the accumulated gel. Place the glass plate with the gel in a fixative or coloring solution to float the gel away from the plate. All steps of gel coloration must be performed at room temperature with gentle agitation in a suitable container (e.g., glass dish or tray for photo development) (e.g., on top of a fixed-rail shaker platform). It is always necessary to wear gloves when staining gels because fingerprints can stain. A permanent record of the stained gel can be obtained by photographing the gel or by placing it on filter paper and drying it in a commercially available desiccator. Caulmers Brilliant Blue R-250 staining is the standard method for protein detection (Allen et al., 1984; Andrews, 1986; H ames, 1981; Wilson, this series). For protein bands to be easily visualized, each band should contain 0.1 to I ug of protein. (1) Preparation of staining solution: Dissolve 0.1 % komas Brilliant Blue R-250 (m " ) in 40 % methanol (V /V ), 10 % acetic acid (V /V ). The dye solution was filtered when the dye was dissolved. The dye solution can be reused. Store at room temperature. (2) Soak the gel for 30 m i n using an excess of staining solution. (3) Decolorize using a large excess of 4 0 % methanol, 1 0 % acetic acid. The decolorizing solution needs to be changed several times until the removal of the background color has been satisfactory. The acidic ethanol solution used in this step does not completely immobilize the proteins in the gel. This can result in the loss of some low molecular mass proteins during staining and decolorization of thin gels. Therefore, permanent immobilization can be achieved by incubating the gel in 40 % methanol (V/V), 10 % trichloroacetic acid (m/V) for I h before immersion in the staining solution. This method, invented by Merril and coworkers, is more than 100-fold more sensitive than the dye staining method (Allenet al., 1984; Merrii et al., 1981, this series), and bands containing 10 to 100 ng of protein are easily visualized. Reagents are available in kits from Bio-Rad Laboratories, Inc. The reaction time varies with the thickness of the gel. (1) Fixation of proteins in the gel in 400 m L of 40% methanol, 1 0 % acetic acid (V /V ) (or 40 % methanol, 1 0 % trichloroacetic acid) can be 30 m i n to overnight. (2) Fix the gel twice in 400 m L of 10 % ethanol, 5 % acetic acid (V /V ) for 15 to 30 m i n . (3) Soak the gel in 200 mL of fresh oxidizer solution (0.0034 ml/L potassium dichromate, 0.0032 ml/L nitric acid) for 3 to 10 m i n . (4) Wash the gel 3 or 4 times with 400 mL of water for 5 to 10 m i n each time until the yellow color is removed. (5) Soak the gel in 200 mL of fresh silver reagent (0-012 m o l /L silver nitrate) for 15 to 30 m i n . (6) Wash the gel with 400 m L of water for 1 to 2 min. (7) Wash the gel with developer (0.28 mol/L sodium carbonate, 1.85% polyformaldehyde) for 1 m in. (8) Replace the gel with fresh developer and incubate for 5 mM. (9) Replace the developer again and wait for development until satisfactory staining results are obtained. (10) Stop developing with 5 % acetic acid (V/V). Vertical streaks and sample-independent bands are sometimes seen in the 50-70 kDa region of silver-stained gels. These artifacts are caused by reduction reactions of contaminants inadvertently introduced into the sample (Pchs 1983). These artifacts can be eliminated by treating the sample with SDS-reducing buffer and then adding excess iodoacetamide. The rapid, single-step SD^PAGE gel staining method can be accomplished by immersing the gel in copper chloride (Leeetal., 1987). The staining result, i.e., the negatively stained image in the electropherogram, is intermediate in sensitivity between that of komas blue and silver staining. (1) Wash the gel briefly with water. (2) Soak the gel in 0.3 mol/L CuCl2 for 5 min. (3) Wash the gel with water for 2~3 min. This method results in a negatively stained gel, i.e., a clear protein band on an opaque blue-green background. The protein bands are easily visualized and the gel can be developed on a black surface. In this method, the protein is not permanently immobilized and can be quantitatively eluted after chelation of copper (Lee et al., I987). The electrophoretic pattern is lost when the copper-stained gel becomes dry. Therefore, they must be photographed promptly, restained in Cauloblue or stored in water. Protein molecular mass standard mixtures can be used for gel calibration. The p a g e standards are mixtures of proteins of precisely known molecular mass mixed by uniform staining. They are in different molecular mass ranges. Prior to electrophoresis the standard concentrate reserve is diluted in sample buffer and treated in the same manner as the sample proteins. These proteins are used as reference standards for molecular mass testing. Recently pre-stained SDS--PAGE protein molecular mass standards have come into use. The combination of dye molecules with protein mass standards results in large and unpredictable changes in their molecular mass, so they cannot be used for molecular mass assays. However, pre-stained protein mass standards are very useful in tracking the electrophoretic process and are valuable in evaluating the efficiency of protein transfer during gel blotting. Determination of the molecular mass of a protein can be accomplished by comparing its mobility to a number of molecular mass standards for proteins of known molecular mass. After gel electrophoresis is completed and before staining, the position of the bromophenol blue tracer dye is marked to identify the front of the electrophoretic ionic interface. These can be accomplished by making incisions at the edge of the gel or by inserting a needle soaked in India ink into the gel at the staining front. After staining, measure the migration distance of each protein (protein standard or unknown protein) from the top of the separation gel. The migration distance of each protein is divided by the migration distance of the tracking dye to obtain a normalized migration distance called the relative migration of the protein (relative to the staining front), usually expressed in wind. Create a (half) logarithmic plot of the molecular mass of the protein standard as a function of value. Note that the pattern is slightly S-shaped. Unknown molecular masses can be estimated by linear regression analysis or interpolation by plotting log MT versus Rt as long as they are not at the limit of the molecular mass range. It is important to remember that the molecular masses derived from SDS--PAGE are those of the polypeptide subunits, not those of the naturally occurring oligomeric proteins. The most satisfactory method of recovering SDS PAGE separated proteins for further study is to excise the bands from the gel and extract the proteins. Many attempts have been made to design continuous elution devices suitable for routine protein purification in which the bands appearing at the bottom of the electrophoresis gel are carried away by a slip collector. The scarcity of preparative gel devices demonstrates the lack of success in developing useful devices that are generalized. Ideally, preparative gel electrophoresis should be able to produce high levels of milligrams or even grams of protein, allowing the protein to be recovered purely from the isolates of the corresponding analytical gels. In general, however, band distortion and poor elution limit the resolution achievable with most instruments, making them only effective when used with relatively simple protein mixtures. The difficulties in scaling up gel electrophoresis to the preparative level result in bulky equipment and the need for a great deal of specialized expertise in the pursuit of the best results. For this reason, proteins are generally obtained by analytical gel separation (Harrington, this book). Gels used for the purpose of protein separation can be prepared using special preparative tooth combs (A n d r e w s , l986; C h ra m b ach , 1985). These wide sample well combs span the entire width of the gel and often provide a separate narrow reference well for protein standards. The maximum sample uptake of a gel ultimately depends on how well the protein of interest is separated from neighboring proteins in the sample mixture. Since the bands broaden as the sample volume increases, the corresponding loss of resolution in the separation eventually becomes unacceptable as the sample volume increases. Compared to analytical gel Copper staining is a desirable method for visualizing bands in preparative SDS--PAGE because it does not require a fixative. The target bands are excised from the gel and decolorized by immersion in a solution of 0-25 m o l / L EDTA, 0.25 m o l / L Tris-HCl (p H 9), which is changed three times (10 m i n each time). After decolorization, the gel sections were immersed in the appropriate elution buffer. Proteins are usually extracted from the soaked gel slices either by simple diffusion into the appropriate buffer or by solubilization of the gel (A n d r e w s ,l986 ;Harrington, this book). In the latter method, a cross-linking agent different from bisacrylamide is polymerized into the gel (Allen et al, ,1984 ;A n d r e w s , 1986). For example, gels crosslinked with N ,N '-bis(acrylyl)cystamine (B A C ) are soluble in 2-mercaptoethanol or dithiothreitol, and gels crosslinked with N ,A ^- dihydroxyethylenebis(acrylamide) are soluble in 2-mercaptoethanol or dithiothreitol. dihydroxyethylenebisacrylamide (D H E -B A ) and JV,N -tartaric acid bis(propylidene)clownamine (] V,N 7 ~diallyltartardiamide, D A T D ) intersections make the gel soluble in periodate. Once the gel is soluble, proteins must be separated from the excess gel medium by gel filtration or ion exchange chromatography. Electrophoretic elution is an effective method that can recover proteins from gel sections. In the simplest version of this method, equipment for gel column electrophoresis is used so that proteins are electrophoretically removed from the gel slice and into the dialysis bag. The equipment allows rapid recovery of small amounts of protein, and in most cases yields are higher than 70%. Elution takes about 3 h in 0-025 m o l / L Tris, 0.192 m o l / L glycine, 0.0% SDS (p Hg), 0.5 m o l / L Glycine, and 0.5 m o l / L Glycine. l% S D S ( p H 8. 3) in a solution (standard S D S ^ P A G E electrophoresis buffer) at 10 m A / tube. S D S can be removed from eluted samples by dialysis or ion exchange chromatography (F u r t h , 1980). For more product details, please visit Aladdin Scientific website.
Keep the acrylamide stock solution in a dark colored bottle at 4° C for a period of not more than 1 month. WARNING: Acrylamide monomer is a neurotoxin. Avoid aspiration of acrylamide powder, do not pipette acrylamide solutions by mouth, and wear gloves when handling acrylamide powder or solutions containing acrylamide. Dispose of unused acrylamide by adding bisacrylamide (if bisacrylamide is not present in the acrylamide solution) to induce polymerization and discard the solidified gel.
Place under vacuum for at least 15 min to remove air bubbles from the solution (e.g., in a bell glass or desiccator).
After 1 h, the cumulus can be poured in (Bio-Rad Lab, Bull. No. 1156). The unused monomer is polymerized in a beaker and the gel is discarded.
In the presence of SDS, this compact conformation can be maintained to a greater or lesser extent. Thus some SDS proteins without 2-mercaptoethanol migrate faster than proteins with reductants that cause structural stretching during electrophoresis. Proteins often exhibit unique responses to reducing agents, so much can be learned by comparing SDS-PAGE with and without 2-mercaptoethanol (Marshall, I984).
Compared with analytical gels, it is permissible to sample 10 to 50 times more protein per unit of cross-section in preparative gels. As a result, tens of milligrams of protein can be recovered on some of the large plate gels.