Protein Characterization by Electrophoresis

EXPERIMENT NO. 15 PROTEIN CHARACTERIZATION BY ELECTROPHORESIS Abstract The molecular(a)(a) weights of protein extracts were assessed by atomic number 11 dodecyl sulfate-polyacrylamide gelatineatinatine dielectrolysis (SDS-PAGE). devil inureds of four protein samples, standard bovine serum albumin (BSA), invertase, egg albumin, and casein, were alert wizard set containing ? -mercaptoethanol (BME) duration the anformer(a)(prenominal) did not. These were because analyzed through SDS-PAGE with 12. 5% firmness gel, prepared apply 2 M Tris-HCl at pH 8. 8 and stacking gel, prepared victimization 0. 0625 M Tris-HCl at pH 6. . Results showed multiple solidifyings lay on the upper half of the gel, which call downed heterogeneity of the variety show and that the samples were profound molecules. Introduction Proteins are biologic macread-only memoryolecules imperturbable of bingle or more(prenominal) polypeptides, which are polymers of amino group acids. Structural ly diverse, these molecules in addition serve a unnumberable of functions from enzymes, which are the biological catalysts of many physiological re achieves, to comp singlents that go along the structural integrity and organization of cells (Pratt and Cornelly, 2011).Because of this, it has been a continual effort among chemists to extract and isolate proteins to determine the mechanisms by which they act and produce the conclusions of their receptions. Further knowledge of their biological action could translate into the disc everyplacey of many resources that could palliate humans and other species daily lives. electrophoresis is an analytical tool through which one can understand the case of charged molecules in an galvanising field. legion(predicate) modern electrophoretic techniques use a polymerized gel-like hyaloplasm as a support strong suit.The molecules migration is dependent on the applied electric field, the rigid, mazelike intercellular substance of the gel support, and their surface, shape, charge, and chemical substance composition. The movement of a charged molecule in an electric field is utilisen by v=Eq? f (1) where v is the velocity of the molecule, E is the electric field magnitude, q is the net charge of the molecule, and f is a frictional coefficient dependent on plentitude and shape of the molecule. Hence, it is observed that under a changeless electric field magnitude, the movement is dependent on the charge-to-mass ratio of the molecule.Since each molecule is expected to ca-ca unique charges and sizes, their mobility under the electric field would alike be divers(prenominal). Gels use in electrophoresis with antithetic pore size may be produced by using different assiduitys of cross-linking factors. Polyacrylamide gel electrophoresis (PAGE) allows intensify re firmness of sample components due to breakup establish on molecular sieving and electrophoretic mobility. Because of the presence of a continuous ne devi lrk of pores in the gel, large molecules do not move easily through the medium compared to modester ones.deuce types of gels are used the resolving power and stacking gels, each having different concentrations of acrylamide and of different pH and bonce strengths. The denaturants sodium dodecyl sulfate (SDS), a detergent, and ? -mercaptoethanol (BME), a reducing agent, are frequently used in PAGE. The action of these two denaturating agents cause the production of polypeptide chains of unalterable charge-to-mass ratios and uni found shapes due to the SDS molecules spine with the hydrophobic regions of the modify polypeptide and masking the native charge of the protein by its contradict charge.This restriction, coupled with the fact that mobility of the SDS-protein complexes are based on molecular size, forms the basis of the electrophoretic determination of rectitude and molecular weight (Boyer, 1993). This experiment will utilize SDS-PAGE to assess the molecular weights o f the extracted proteins invertase, albumin, and casein, along with standard bovine serum albumin. The effect of the presence of ? -mercaptoethanol was also investigated. ruleology With the glass plates clean, the gel apparatus was first set up with the comb inserted between the glass plates.It was do genuine of that the set-up would not leak by allowing a little amount of distilled water to enter it, which was get rid of laterwards. A mark, one centimeter below the odontiasis of the comb, was placed on the glass plate. The resolving gel, at 12. 5% gel, was indeed prepared in an Erlenmeyer flask. employ a micropipette, 1450 ? L of 40% conduct acrylamide, 775 ? L of 2% stock bisacrylamide, 875 ? L of 2 M Tris-HCl at pH 8. 8, and 1500 ? L of distilled water were metrical and mixed in the Erlenmeyer flask. after(prenominal)wards, 47 ? L of 10% sodium dodecyl sulfate (SDS) and 40 ?L of fresh prepared 10% ammonium persulfate (APS) were added to the mix. Then, ten microlite rs (10 ? L) of tetramethylethylenediamine (TEMED) was added and, after blend it by swirling not more than troika times, the mixture was poured into the gel apparatus with the aid of a micropipette up to the mark. The gel was then all all oerlaid with a olive-sized amount of isobutanol-water mixture onward it would start to harden. After the gel has all polymerized, the isobutanol mixture was charterd from the apparatus. Two cope withs of two resolving gels were prepared as one pair would be used for samples containing ? mercaptoethanol and another pair for those that do not contain the said chemical. The stacking gel was prepared by taking 265 ? L of 40% stock acrylamide, 140 ? L of 2% stock bisacrylamide, 350 ? L of 0. 625 M Tris-HCl at pH 6. 8, and 940 ? L of distilled water and intermixture all four in an Erlenmeyer flask. Afterwards, 25 ? L of 10% SDS and 60 ? L of 10% APS were added to the one in the flask. Immediately before the solution was added, 5 ? L of TEMED was added to it and swirled not more than three times, similar to that done with the resolving gel.This mixture was then rapidly transferred by a micropipette over the resolving gel and, after placing the comb over it, left to harden. The samples were prepared by getting one C ? L of the protein sample, 20 ? L of distilled water, and 80 ? L of freightage buffer with ? -mercaptoethanol in plastic tubes for the electrophoresis of the samples containing ? -mercaptoethanol. For those samples not containing the latter reagent, 80 ? L of the loading buffer was added. The same influence was done for ascorbic acid ? L of bovine serum albumin.These were then placed in a boiling water tubful for 10 minutes after which these were immediately immersed in an ice water bath for 3 minutes. The protein samples used were invertase, albumin, and casein. The loading buffer was prepared by mixing 2. 5 mL of 10% SDS, 2. 5 mL of 0. 625 M Tris-HCl at pH 6. 8, 2. 5 mL of 10% glycerol, and 5. 0 g of 0. 0 2% bromophenol blue, and diluting to 25 mL with distilled deionized water. Eight tubes were done all-in-all. The gel slabs were then placed in the gel chamber. The gel chambers were then filled with gel running buffer, making sure that the gel was completely immersed.This buffer was prepared by mixing 3. 0 g Tris base, 14. 4 g glycine and 1. 0 g SDS, and diluting to 1 L with distilled deionized water.. The set-up was then placed on a aim surface. At this point, the comb was removed in one fluid motion to ensure that the wells would incur straight edges. Ten microliters (10 ? L) of the samples with ? -mercaptoethanol was loaded into the wells using a micropipette. With the potentiality set at ascorbic acid V and the protective electrode covering placed over the set-up, the gel was run until the dye reaches a direct of 1 cm above the bottom of the gel slab.This was done again for those samples without the ? -mercaptoethanol. After the gels have been run, the gel slabs were transf erred from the glass plates to a flat-bottom container where a small amount of staining solution was added until the slabs were completely immersed. This solution was prepared by mixing 50 mL of methyl alcohol, 10 mL of glacial acetic acid, and 0. 25 mg of Coomassie Brilliant Blue R250, and diluting to 100 mL with distilled deionized water. After that, the downplay staining was removed by several washings of destaining solution.This solution was prepared by mixing 25 mL of 95% ethanol and 5 mL of glacial acetic acid, and diluting to 100 mL with distilled deionized water. Results and Discussion Polyacrylamide Gel Electrophoresis (PAGE) served as an strong tool in the characterization of protein standards and extracts because of the gels high resolving power for molecules up to 106 Da, try-on of larger sized samples, an inert enough matrix with respect to the migrating entities, and physical stability of the matrix (Boyer, 1993).Polyacrylamide gels were prepared by the catalyzed a nd cross-linked polymerization of the acrylamide-bisacrylamide mixture. The polymerization reaction was facilitated by ammonium persulfate (APS), the polymerizing agent, due to its inherent instability and, hence, its endeavor to disintegrate and to give rise to molecules initiating these polymerization. Tetramethylethylenediamine (TEMED) was introduced to catalyze the decay of APS. Figure 1 presents the general equation for the polymerization reaction of the acrylamide-bisacrylamide mixture (Encor Biotechnology, Inc. , 2011). Figure 1.The polymerization reaction of the Acrylamide-bisacrylamide in the presence of ammonium persulfate and TEMED as the polymerizing agent and the catalyst respectively (Thermo Scientific, Inc. , 2011) Polymerization proceeded with the opening of an acrylamide in two ways bond, allowing it to react with another acrylamide to produce a elongate polyacrylamide. Cross links were generated through the incorporation of bisacrylamide into the running(a) po lyacrylamides. Since molecular group O would react with the free chemical group sulfate ions (SO42-) in that locationby inhibiting polymerization, degassing was necessary.Furthermore, the tendency of molecular oxygen to react with SO42- would also be the reason wherefore it would be necessary for PAGE gels to be poured into tubes or between glass plates instead of horizontal apparatuses. However, the degassing step was not done due to the unavailability of a degassing chamber. Isobutanol was added on top of the gel to also anticipate the entry and accumulation of O2 (Encor Biotechnology, Inc. , 2011). Gel pore size is inversely proportional to the concentration of acrylamide.Therefore, to generate a broad and efficient range of protein separation, a discontinuous gel system was formulated, having a low acrylamide cognitive contented on top and a high acrylamide content at the bottom. The capability of Tris-HCl to facilitate the propagation of electric current through the ma trix qualified it as an appropriate loading buffer. It allowed the proteins to be drawn by the current through the sieving matrix slab (Thermo Scientific, Inc. , 2011). The polyacrylamide gel electrophoreses set-up had three important features. First, a stacking gel was cast over a resolving gel.Second, the two gel layers had different ionic strengths and pH. third gear, the stacking gel had a lower acrylamide concentration and a lower pH. These conditions allowed the protein molecules to first concentrate into a tight band before entering the resolving solution. In this experiment in particular, the charge of the protein was kept uniform all throughout using sodium dodecyl sulfate (SDS), a powerful detergent that would modify the protein and would leave it evenly negatively charged. Also, ? -mercaptoethanol was added to cleave the disulfide bonds, enforcing completely disrupted secondary, tertiary, and quarternary structures.Prior to the loading of the sample, the discontinuous gel system was immersed in a glycine-Tris buffer prepared at pH 8. 8. At this pH, the two form of glycine its Zwitterion ion and glycinate would exist in sense of equilibrium. H3N+CH2COO- H2NCH2COO- + H+(2) When the voltage was turned on, the entry of buffer ions (glycinate and H+) to the stacking gel (pH 6. 8) shifted the equilibrium to the left, increasing the concentration of glycines Zwitterion ion, which would have a zero net charge, and therefore, would be electrophoretically immobile. Since the protein molecules would still be anionic at pH 6. , they would replace the nonmobile glycine molecules in order to carry on the current running. As such, the relative mobilities of the ions in the stacking gel would be Tris base protein sample glycinate. Furthermore, the thin band observed in the upper gel would very pertain to the protein molecules sandwiched between the Tris-base and the glycinate ions. The resolving gel, on the other hand, had a pH of 8. 8. When the ionic fr ont reached it, the equilibrium of glycine species shifted to the right. The increase in pH and reducing in pore size retarded the movement of proteins and rendered the glycinate ions greater mobility.The relative rates of movement then became Tris-base glycinate ions protein samples. From there, it was the mass of the protein molecules that governed their mobility and thus identified them (Boyer, 1993). For qualitative epitome of results, the Coomassie brilliant blue dye (R-250), being the about popular staining reagent for the electrophoresis of protein samples, was used. Its mechanism of binding to the basic and hydrophobic groups of proteins manifested in the dull, reddish-brown to graphic blue color change of the solutions.The staining method was started with the water wash of the gel cast to remove the electrophoresis buffers from the matrix. The matrix was then washed with methanol followed by glacial acetic acid to hinder the diffusion of protein bands form the matrix . The treatment with the dye followed. Lastly, destaining measures were use to get rid of excess dye from the background gel matrix. This would allow a clear visual image of the bands that had formed (Thermo Scientific, Inc. , 2011). Figures 2 and 3 are photographs of the two gels after incubation and subjection to the dye. Figure 2. image of 1st gelFigure 3. Photograph of second gel In figure 2, multiple bands existed. This could suggest that the samples had other components. These could come in the form of other proteins, contaminants, or other impurities. Nonetheless, any of these possibilities suggest one thing the sample is not pure although there are occasional times when homogeneous samples result to multiple bands due to degradation during the electrophoresis procedure (Boyer, 1993). Also, the identity of the proteins could have been fixed if there were standards or markings to compare these bands with. However, there were none.The only information that could be extracte d from the photographs could be that the proteins in the samples were heavy that they were only set on the upper half of the gel. Conclusion The separation of biomolecules according to charge, size, and shape through electrophoresis could give significant information such as the purity, molecular weight, and, hence, the identity of the biomolecule. In this experiment, the multiple bands produced in the gel set-ups suggested that the samples were heterogeneous. Their location in the gel suggested that the proteins were relatively heavy ones. To gain more valuable information rom these data, it is recommended that a set of standard solutions be also run on the gel so that they could be used as references for the identification of the proteins in the samples. Also, the proteins exact molecular size could be determined by preparing a calibration curve from a set of standard solutions of proteins, with of course, known concentration. The curve should be a plot of the logarithm of the mo lecular weight of the protein versus its mobility in the gel matrix. From this curve, the molecular weight of the protein in the sample solutions could be extrapolated. References 1. Boyer, Rodney.Modern Experimental Biochemistry. Third Edition. San Francisco, USA Benjamin/Cummings, 1993. Scribd. Web. 29 November 2011. 2. Encor Biotechnology, Inc. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Encor Biotechnology, Inc. Protocols. Encor Biotechnology, Inc. , 2011. Web. 30 November 2011 http//www. encorbio. com/protocols/SDS-PAGE. htm. 3. Thermo Scientific, Inc. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Thermo Scientific, Inc. Protein Methods Library. Thermo Scientific, Inc. , 2011. Web. 30 November 2011 http//www. piercenet. com/browse. cfm? fldID=21518847-2D72-475F-A5B9-B236EC5B641E .