Protein Extraction and Gel Electrophoresis - Lab Report Example

 Proteomics and Gel Electrophoresis Abstract Proteomic is generally defined as the direct analysis of proteins in terms of their presence and relative abundance. Gel electrophoresis is a significant methodology employed for extraction of proteins in proteome analysis. The following is a report on an experiment conducted to determine the concentration of proteins in two samples using the Bradford assay and separating the proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It involved three steps, that is, protein extraction, protein quantitation, and gel electrophoresis of two samples. The results obtained showed that the samples both contained 20% protein concentrations. However, during gel electrophoresis, no stain was observed and thus no protein bands could be seen. The possible reasons why the proteins were not separated are discussed. Proteomics and Gel Electrophoresis Proteomic is generally defined as the direct analysis of proteins in terms of their presence and relative abundance. Gel electrophoresis is a significant methodology employed for extraction of proteins in proteome analysis. The most commonly used technique in gel electrophoresis is Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE technique separates proteins based on their capability to move within an electrical current. This ability is a function of the molecular weight of their polypeptide chains. The SDS-PAGE technique achieves this by adding sodium dodecyl sulfate detergent so as to remove secondary and tertiary protein structures and also so that the proteins are maintained as polypeptide chains. The sodium dodecyl sulfate coats the proteins in proportion to their molecular weight and then confers the same negative electrical charge across all proteins in the sample. The rate of migration of a polypeptide in SDS-PAGE is inversely proportional to the logarithm of its molecular weight. This means that the larger the polypeptide, the slower it migrates in a gel. In SDS-PAGE, the molecular weight is determined by comparing the migration of protein spots to the migration of standards. Plots of log molecular weight versus the migration distance are reasonably linear (Dennison, 2003). The proteins separated by SDS-PAGE are often recovered in a procedure that involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing the sodium dodecyl sulfate from the eluted sample, and finally renaturing the protein for subsequent analysis. Proteins that are eluted from gels are used in varied downstream applications successfully, such as protein chemistry, determination of amino acid composition, identification of polypeptides that correspond to specific enzyme activity, and other purposes. The analysis of protein concentrations is a significant assay in biochemistry research. The Bradford assay is one of the most widely used methods to determine concentrations of protein, relative to a standard. The technique is based on the formation of a complex between proteins in solution and the dye, Brilliant Blue G. This assay is commended for overall use, particularly for assessing concentrations of proteins for gel electrophoresis. It is based on observations that absorbance maximum for acidic mixtures of Coomassie Brilliant Blue G-250 that do shift from 465 nm up to 595 nm at a time when protein binding occurs. The assay is effective because of the extermination coefficient of the albumin-dye complex solution is usually constant over a range of 10-fold concentration (Westermeier, Naven & Höpker, 2008). The dye reacts mainly with arginine residues but less with histidine, lysine, tryptophan, tyrosine, and phenylalanine residues. Seemingly, this examination is not all that perfect for acidic or basic proteins. However, it is somewhat sensitive to the bovine serum albumin, even more than most proteins, by a factor of two. Gamma globulin (IgG) is the protein standard of preference. The objective of this experiment was to determine the concentration of proteins in two samples using the Bradford assay and extracting the proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Materials and Method Materials The following materials were used to perform the experiment: Protein extraction Buffer: RIPA buffer (Thermo Inc.) Phosphate buffer Protein quantitation 1mg/mL Albumin Bradford reagent Distilled water 96-well plate Gel electrophoresis Stacking gel and separating gel Loading dye/loading buffer Running buffer Method In order to extract the proteins, cell suspensions of Samples A and B were prepared by centrifuging 1500ul of each for 5 minutes. Their solutions were discarded and the platelets retained. 1000ul of ice-cold PBS was then added to the cuvettes with the platelets and centrifuged again for 15 minutes. 200ul of RIPA, which was the lysis buffer, was then added to both Sample A and Sample B and the samples centrifuged at 14,000g for 20 minutes to remove the cell debris. The supernatant was retained as the sample solution. To quantify the proteins present in the sample solutions, the Bradford assay was employed. 1X Bradford reagent was diluted with distilled water in the ratio of 1:2. The albumin standard solution and the sample solutions were then prepared. 200ul diluted Bradford reagent, 5ul of the albumin standard, and 5ul of Sample A and B was added into the 96-well plate and the contents mixed. The absorbance at 595nm was then measured with a spectrophotometer and the data recorded. Next, 20mg of the protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. First, the SDS PAGE gel was prepared. The protein sample was then mixed with loading dye in 5:1 ratio and heated in boiling water for 10 minutes. The sample was pipetted to sample wells, the electrophoresis run, and the image of the gel recorded. Results Table 1:1 Protein Absorbance at 595nm Sample Concentration Albumin Standard Sample Alb. 0 D.W 100 Alb. 25% D.W 75 Alb. 50% D.W 50 Alb. 75% D.W 25 Alb. 100% D.W. 0 A B Abs. at 595nm 0.167 0.316 0.320 0.451 0.537 0.261 0.263 The table above showed the absorbance of the albumin standard and the sample solutions at 595nm. Deducing from it, Sample A and Sample B both seem to have the same concentration of proteins. In comparison to the albumin standard, the samples had a 20% protein concentration. During SDS-PAGE gel electrophoresis, the gel image did not show any results as there was no stain observed. Discussion The Bradford assay assessed that there was about 20% concentration of proteins in both sample solutions. However, no stain was observed when the sample solutions were run during the electrophoresis. The protein bands were not seen properly and therefore, they could not be identified. There are several reasons why this could have happened. First, it could be that the Coomassie Brilliant Blue G-250 stain in the Bradford reagent was not sensitive enough. To rectify this, the gel can be rinsed and then silver stained. Another possibility is that the protein loaded on the gel was not enough. For Coomassie Brilliant Blue G-250 stained gels, 0.5 micrograms of protein are needed for each protein band to be sufficiently stained. A third reason why there was no stain observed in the experiment is that the volume of Coomassie Brilliant Blue G-250 in the Bradford reagent could have been too little. To solve this, the volume of the staining solution can be increased so as to dilute the SDS that is present in the gel. Use of a more concentrated staining solution and staining for too long can be another possibility for the observed results. Lastly, the concentration of methanol, which strips the SDS from proteins, could be checked and increased in necessary (Gerstein, 2001). As with most scientific methods, gel electrophoresis has its limitations in separating proteins. One of its limitations is that the concentration of acrylamide determines the cut-off at the low end. All proteins below a minimum molecular weight run at the same pace as the tracking dye. They do not separate from each other and are therefore not resolved at all. The second limitation is that the relationship between relative mobility and mass is logarithmic. Resolution of individual bands tends to diminish toward the top of a gel, so that with the more dense gels multiple bands may appear to merge into a single band. Usually, the top 10% or more of a gel is unusable. Conclusion In order to characterize all proteins in a sample, more than one gel should be run. That is a low-density gel that would resolve larger proteins while cutting off smaller proteins, and a higher density gel that would reveal the smaller proteins while compressing and distorting larger proteins. In other words, high-density gels are used to study proteins of relatively low molecular weight and low-density gels are used to study proteins of high molecular weight. References Dennison, C. (2003). A guide to protein isolation. Dordrecht [u.a.: Kluwer Academic Publishers. Gerstein, A. S. (2001). Molecular Biology Problem Solver: A Laboratory Guide. Hoboken, NJ: John Wiley & Sons. Westermeier, R., Naven, T., & Höpker, H.-R. (2008). Proteomics in practice: A guide to successful experimental design. Weinheim: Wiley-VCH. Read More