Protein elution experiments in gels
Protein elution experiments in gels
SDS-PAGE has proven to be an extremely useful analytical method in determining the number and size of peptides in a sample. It also has the ability to separate many individual proteins of varying sizes if used skillfully. It was hoped by many in the early days of gel electrophoresis that the high resolution properties of the gel could be utilized to obtain small amounts of pure protein.
Operation method
Protein elution experiments in gels Move I. Elution of proteins from gels by diffusion An early method of removing SDS from proteins was published by Weber and Kutter (1971), but when used for trace amounts of proteins, this method is both cumbersome and results in the loss of most of the protein. More early methods in this area have been published (HagerandBurgess,1980), butbearsrevisiting. The basic steps include gel electrophoresis itself, localization of the target proteins in the gel, elution of the proteins from the gel, removal of the SDS and reconstitution of the proteins for subsequent study or use. In the closest approximation to the standard, any of the various gel formulations can be used. There are some key considerations to be made. The polymerization of polyacrylamide gels involves the production of free radicals to drive the polymerization reaction. Therefore, it is important to wait at least 12 h while filling the gel to ensure that the polymerization is complete. Since the reaction initially creates an oxidizing environment, you need to take precautions to protect the protein from oxidation. This is easily accomplished by adding cheap, low molecular mass, internal carrier proteins (e.g., β-lactoglobulin) that run ahead of most proteins during electrophoresis to the samples to be loaded; these carrier proteins consume the oxidizing agent remaining in the gel. It is also necessary to add anionic sulfhydryl complexes (we use 0.1 mmol/L sodium thioglycolate) to the buffer in the upper pool; they will cross the gel during electrophoresis, destroying any potentially oxidizing substances and remaining free radicals in the gel. The reaction of cysteine with free acrylamide produces cysteine-S-acrylamide, which can also be reduced by the presence of sulfhydryl groups in the gel (Chiarietal., 1992). High-quality SDS containing only the dodecyl form (C12) should be used because impure SDS may contain varying amounts of C14 or C16, which bind proteins more strongly than C12 and are very difficult to remove (KunitaniandKresin, 1989). Once gel electrophoresis has begun, it is necessary to determine the location in the gel that contains the target bands or bands of a specific molecular mass. Of course, if you don't know the size of your enzyme bands, you can cut the gel into sections, elute the proteins from each section, and analyze all sections for enzyme activity. Initially, we determined the bands we were interested in by soaking the gel in cold 0.25 mol/L KCl for 5 min, and then destaining it ih cold distilled water. We now know that you can stain the gel by almost any convenient method, including the zinc-imidazole (CastelIanos-SerraandHardy, 2001) method; or by using one of the newer, very sensitive fluorescent protein dyes such as SYPRORuby (see Chapter 31 for details). You can even use a Khomas Brilliant Blue dye. In one instance, we successfully reproduced a strip that had been stained with Kaomas Brilliant Blue, color-coated, dried, and stored for 10 years! Another method is to electrophoresis colored standard molecular mass proteins (available from many companies) on gel flanking lanes. This method guides us to cut out specific parts of the gel based on molecular mass information. Often overlooked, this simple step is inexpensive, effective, and allows for the simultaneous processing of multiple samples. The typical scheme described below is based on a modification of the method described by Hager and Burgess (1980). (1) The SDS gel is washed with cold distilled water and stained with ice-cold 0.25 mol,7LKCl and 1 mmol/L DTT for 5 min to determine the position of the bands. The gels were rinsed with double-distilled water and decolorized with cold double-distilled water and Immol/LDTT for 10~60 min. Alternatively, a gel lane can be divided into a number of 3-5 mm long sections, and each section can be placed in a test tube and washed as described above. (2) Crush the gel sheet in ImL elution buffer. Initially we used a 3.5 mL siliconized Pyrex glass test tube (10 mmX75 mm) and a small Teflon mashing rod (Kontes, K886001size19), but nowadays a 0.3 mL elution buffer, 1.5 mL polypropylene centrifuge tubes, and disposable polypropylene mashing rods (e.g., Kontes Mashing Rods. K749521-1500). The elution buffer was 50 mmol/LpH7.9 Tris, 0.1 mmol/LDTA, Immol/LDTT, 0.15 mol/L NaCl, 25-100ug/mL BSA (without addition) and 0.1% SDS. (3)-Once the gel is crushed, small gel fragments need to be incubated in a rotator for 1~8 h to allow passive diffusion. In the method of Hager and Burgess (1980), the elution kinetics showed that the elution half-life of 36 kDa peptides was less than 30 min and that of 150 kDa peptides was 1?I.5 h in 8.75% polyacrylamide gel medium (4 h and 16-24 h for complete elution, respectively). (4) The mixture was centrifuged in a centrifuge at maximum speed for 2 min to precipitate the broken gel. The supernatant (protein eluate) is then transferred to a clean microcentrifuge tube. Since elution efficiency is highest with 0.1% SDS in the elution buffer, SDS must be removed after elution, and acetone precipitation is the most efficient method, not only removing SDS, but also concentrating the protein. A typical procedure based on Hager and Burgess (1980) is described below. (1) Add 4 times the volume of cold acetone (-20°C) to the protein eluate and allow the sample to precipitate in a dry ice-ethanol bath for 30 min. Since the sample freezes in the dry ice-ethanol ice bath, thaw the sample briefly in an ice-water bath before centrifugation. (2) Centrifuge at maximum centrifugation rate for 5 min. remove and discard the supernatant. In a test experiment using 0.16 radioactive RNA polymerase, we found that when the concentration of carrier BSA in the elution buffer was 0ug/mL, 15/ng/mL, and 100pg/mL, the corresponding recoveries of the polymerase were 71%, 90%, and 99% (HagerandBurgess, 1980).(3) Tests showed that more than 99.9% of the SDS remained in the acetone supernatant. SDS remains in the acetone supernatant. The precipitate can be washed by using ImL of ice-cold 80% acetone and centrifuged again to remove residual SDS. (1) The acetone precipitate should be dried for 10 min. (2) The precipitate is dissolved and denatured in dilution buffer containing 206 mol/L guanidine hydrochloride (GuHCl) [Dilution buffer is 50 mmol/LTris (pH 7~9), 20% glycerol, 0.Immol/LEDTA, 1 mmol/LDTT, 0.15 mol/LNaCl, 20?100 fxg/mL BSA (may be left out) and 0.1% SDS were solubilized and denatured. Dissolution occurred within 20 min at room temperature. (3) Rapidly dilute the solubilized proteins 50-fold by adding I.0 mL of dilution buffer, and denature for 1-12 h at room temperature. (4) Analyze the reconstituted proteins by appropriate analytical methods. Preliminary experiments on the target enzyme were carried out to observe its ability to recover in 6 mol/L and diluted guanidine hydrochloride, which can be used to test the recovery of the specified proteins. If the recovery is good, the recovery of activity is tested by denaturation in SDS elution buffer, acetone precipitation, solubilization in 6mol/L guanidine hydrochloride and dilution. Although the diffusion elution method is very versatile, it cannot be applied to all enzymes. It cannot be used if the enzyme activity is dependent on two or more peptides of different sizes, or if separable cofactors such as heme (which leaves the catalytic protein during SDS gel electrophoresis) are required. The method is also difficult to use if the protein has post-translational modifications that may affect refolding, such as glycosylation or proteolytic processing, or if there are essential disulfides that are difficult to improve. Despite these limitations, diffusion elution has successfully denatured many proteins. These include DNA topoisomerases, DNA ligases, bacterial a-factors (Haldenwang et al., 1981; Wiggsetal., 1981), eukaryotic transcription factors, H-RasGTPase, methyl reductase, filament-binding proteins, and protein components of protein/RNA ribonucleases. The method has been used for enzymes from bacteria, yeast, human, Drosophila and many other species. The method can also be used for enzymes containing multiple identical subunits. The method can also be used for proteins containing different subunits if appropriate gel slices are mixed together. This method has been successfully used for proteins containing essential S-S bridges. The clever modifications to the Hager and Burgess method discussed above have been published by Prokipcak et al. (1994). They replaced the SDS susceptibility and acetone precipitation methods with reversed-phase high-performance liquid chromatography (Rp-HPLC) and lyophilization. The proteins were applied to a C4 reversed-phase high-performance liquid chromatograph (RP-HPLC) and eluted with a gradient of 0% to 50% acetonitrile/0.1% TFA. The eluted protein fractions were lyophilized, resuspended in a small volume of 6 mol/L guanidine hydrochloride, diluted to refolding, and analyzed for enzyme activity. The above scheme describes in detail the use of diffusion to elute proteins from gels, and many of the operations should be perfectly suited to electrophoretic elution. Although these methods are more efficient than diffusion methods, they are more expensive and difficult to scale up for multiple gel slices, even though the elution can be accomplished more efficiently in a shorter period of time. The SchleicherandSchuellElutrap and the Bio-RadModel422Electroeluter? are two of the most widely used commercial electrophoretic elution devices. Both devices involve the placement of a gel slice containing a target protein band into the device and the elution of the protein from the gel slice by protein electrophoresis, where the protein passes through a large pore membrane or glass sieve (frit) into a chamber with a small pore membrane that allows only small molecules, or proteins smaller than about 5 kDa, to pass through. The target protein is pipetted out of the chamber and used as required. More detailed instructions for the use of these commercial products can be found in the excellent reviews by Harrington (1990) and Seelert and Krause (2008), as well as in company literature. Another commercial method is the ProteoPLUSelertoeluter. a small screw cap tube is filled with upstream and downstream protein retention membranes that allow an electric current to pass through the tube when it is placed in electrophoresis. After the proteins are eluted from the gel sheet, they can be dialyzed in the same tube for subsequent use. An example of the application of this method is given by Lei et al (2007). The Bio-RadWholeGelEluter can be electrotransferred into 26 sections in a single flat gel. Typically a protein sample is uploaded onto one lane of the gel width for SDS gel electrophoresis.The 26 grooves of the Eluter run parallel to the protein bands. Proteins are transferred out of the gel into the caskets and into the collection box. Another recent development involves the elution of multiple individual fractions of a flat gel into multiwell plates for proteomic analysis (Antaletal., 2007). Protein bands separated by high-resolution gel electrophoresis can be recovered from the gel and used in a large number of applications. Elution of proteins from a gel sheet is accomplished by crushing the gel and diffusing the proteins out of the gel; alternatively, an electric field can be applied to the gel sheet and the eluted proteins can be captured on a suitable membrane-bound device. Sub-micrograms to 100ug of protein can be obtained, which is usually denatured to obtain enzymatic activity. For more product details, please visit Aladdin Scientific website.