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Proteomics Lab Report

The University of North Texas BIOL 3520.507 TA: Neha Iyer STUDENTS

Abstract: Central dogma is the flow of genetic information from DNA to RNA to make proteins. Proteomics is the large scale study of proteins, and in the first part of the experiment, actin and

myosin from five different fish species were analyzed to determine their common ancestry. They were then assessed via western blotting to determine the presence of myosin light chain. The results showed that the yellow fin tuna and the swordfish shared the most protein bands, thus they shared the most common ancestor on the cladogram, and the presence of myosin light chains in each fish sample was detected, confirming the presence of actin and myosin in each fish sample. Introduction: With over fifteen thousand varying species of fish, and an additional 150 species added annually, how do scientists determine these species’ taxonomic classifications? With years of evolution and genetic adaptations, even the smallest changes could create a new classification to occur. When looking at Swordfish, Yellow Fin Tuna, Atlantic Cod Fish, Salmon, and Tilapia, these fish vary drastically, yet, they all derive from a common ancestor. By using protein gel electrophoresis, a technique used to examine muscle proteins, scientists are able to identify similarities and differences in these organisms’ protein profiles. From their molecular data, it is also possible to determine their similarities. Each protein band a fish shares in common with another is a shared trait. A cladogram can be constructed based on the number of protein bands the fish have in common. When two organisms share a common trait, they also share a common ancestor. Actin and myosin, are the primary proteins that make up muscle tissue and are highly conserved across all animal species. Other proteins are more diverse, varying even among closely related species (1). Proteomics is the study of proteins and their environment, and aims to describe each protein in an organism. A proteome is the collection of proteins that make up an organism and vary amongst cells and different organisms. They are constantly changing through various interactions both biologically and environmentally. A specific tool most commonly used in proteomic research is an immuno-detection technique known as Western Blotting. This technique is used to detect and quantify proteins in complex biological samples. Gel electrophoresis and western blotting are used to identify a subunit of a myosin light chain from the different proteins that make up the muscle tissues of fish samples. The myosin light chain proteins will be compared for each sample (1).

During this experiment, protein; specifically actin and myosin, will be extracted from five different species of fish (swordfish, yellow fin tuna, atlantic cod fish, salmon, and tilapia) and tested to determine which species are most closely related. Once the protein is extracted, the concentration of protein is to be determined. The protein samples will then be added to Sodium Dodecyl Sulfate (SDS) and heated. This process denatures the protein samples and allow them to become linear with a negative charge. This in turn allows the protein samples to undergo gel electrophoresis. Using Polyacrylamide gel electrophoresis, or PAGE, proteins will be separated based off their molecular weights. From the gel, a cladogram can be constructed and the common ancestry of the different fish species can be analyzed. For a more quantitative result, a Western Blotting technique will be run and immuno-detection will be used to look for myosin light chains. From the immuno-detection, the molecular mass of each protein sample can be calculated and a standard curve can be constructed. From the data collected, linear relationships with both molecular weight and distance migrated can be formulated (1). From the information collected about each fish sample, each fish is a different species, yet from the FishBase, they share multiple similarities. The Swordfish, Yellow Fin Tuna, and Tilapia are all perciformes, meaning they have the same “order” when discussing taxonomic classifications. This alone, should tell that these three species would most likely share a common ancestry. The perciformes also share a physiological similarity in which they all live in tropical temperatures which are relatively warmer than the cod and salmon. If the cladogram were to be constructed, than the tilapia and the yellow fin tuna would share the most common ancestor because they are both perciformes and they physically share the most qualities. The western blot will also confirm the presence of the myosin light chain because they are all fish, and their muscle movement is regulated by both actin and myosin. Materials & Methods: To begin the experiment, a mini grinder is to be prepared by first labeling each fish sample A-E and then centrifuging the tubes with resin for 20 seconds, and then removing the liquid collected with a pipette. Each tube will then add 500 microliters of PBS Buffer while the fish sample is grinded. Once it is thoroughly grinded, another 500 microliters of PBS Buffer will be added and then the samples will be centrifuged for 5 minutes at 20 degrees celsius. Then the liquid above the pellet should be carefully transferred to a new

tube without disturbing the pellet. The protein samples should then be cleaned by suspending the settled gel and purifying the proteins. By letting the samples run through the column and by centrifugation, each fish sample has been excluded to only pass molecules smaller than the limit. From these samples, their protein concentration is to be determined. By running a series of dilutions, 12 cuvettes will be used, adding 1 milliliter of Bradford Reagent (add to all), 20 microliters of each standard (only to the first 7 respectively), and 20 microliters of each fish protein sample (only add to the last 5 respectively). Incubate at room temperature for 5 minutes and then measure readings in spectrophotometer. After the protein samples have been plotted, a standard curve is to be determined and from it, an R2 value and equation for a line of best fit will determine the concentrations of the samples. Once the protein concentrations have been determined, gel electrophoresis is to occur. Before running electrophoresis, the addition of SDS and laemmli buffer will allow the protein samples to denature and gives the proteins a negative charge. Set up the MiniPROTEAN and add 1x TGS electrophoresis buffer to the chamber. Prepare the cassette and then slide the gel cassette, buffer dam and electrode assembly into the clamping frame. Heat the fish samples along with the actin and myosin and load the samples into the gel. Once the gel has been loaded, electrophorese for 30 minutes and then transfer the gel to a container with 25 ml Bio-safe coomassie blue stain and stain gel for 30 minutes while gently shaking. Analyze the gel and construct a cladogram. For the second portion of the lab, proteins will be separated by polyacrylamide gel electrophoresis. Set up the Mini-PROTEAN and add 1x TGS electrophoresis buffer to the chamber. Prepare the cassette and then slide the gel cassette, buffer dam and electrode assembly into the clamping frame. Heat the fish samples along with the actin and myosin and load the samples into the gel. Once the gel has been loaded, electrophorese for 30 minutes and then transfer the gel and chop the wells off very carefully. Transfer the gel to a plate with the blotting buffer and equilibrate. Soak the fiber pads and nitrocellulose membrane in blotting buffer and make a blotting sandwich: fiber pad, blotting paper, membrane, gel, blotting paper, and fiber pad. Set up the tetra blotting apparatus and fill tank up with blotting buffer. Attach the module and run blot for 30 minutes. Once complete, dismantle and place membrane in 25 milliliters of blocking solution with pre-stained standards facing up. From the western blot, antibodies will be used to detect the proteins by

immuno-detection. Begin by pouring off the blocking solution, and adding 10 milliliters of anti-myosin primary antibody and gently shaking for 10 to 20 minutes. Pour it off, and then rinse the membrane in 50 milliliters of wash buffer and then pour that off. Add 50 milliliters of wash buffer and gently shake for 3 minutes and then pour off. Add 10 milliliters of the secondary antibody and repeat wash buffer steps previously noted. Add 10 milliliters of horseradish peroxidase and gently shake for at least 10 minutes to allow bands to develop. Once bands begin to develop, discard the reagent and rinse the membrane twice in distilled water. Air dry the membrane and store in the dark. Using the membrane, determine the molecular mass of myosin for each sample and construct a standard curve. Analyze the data to determine the distance travelled.

Data & Analysis:

[Figure 1: Initial Cladogram]

[Figure 2: Bradford Assay Standard Curve]

[Table 1: Absorption Rates to Determine Concentration]

[Figure 3: Gel Analysis]

[Figure 4: Other Lab Groups Gel Analysis]

[Figure 5: Standard Curve SDS PAGE]

[Table 2: Protein bands]

[Table 3: Comparison of Protein bands]

[Figure 6: Final Constructed Cladogram]

[Figure 7: Western Blotting and Immunodetection]

[Table 4: Immunodetection] Discussion of Results: Figure 1 is a representation of the initial cladogram constructed before the experiment began. It was constructed solely off prior knowledge, physiological characteristics, and their traits. Figure 2 is the bradford assay standard curve, which was constructed by plotting the

absorbance (y) against the standard concentration (x). The standard concentration was created by performing a serial dilution and the graph constructed was reliable since the correlation coefficient was 0.984, which is very good. This was used to compare the absorption rate of the fish samples. In Table 1, the protein concentration of each fish sample was calculated. This allowed us to determine how much laemmli buffer to each sample for proper dilution. Figure 3 is the direct results of the gel analysis. The gel was skewed to the middle, thus rendering our data void, so Figure 4 was used to calculate our data, based off another groups’ lab result. The skew in the middle of our gel could have occurred due to multiple reasons, however; uneven surface placement is what is believed to have been the cause. In Figure 4, their gel analysis came out clear, so we were able to measure the distance of the bands. In Figure 5, the kaleidoscope lanes was used to develop a standard curve to see the Rf value. This gave us the values of the distance travelled in the ladder. The log of the molecular weight (x) was plotted against the Rf value and the correlation coefficient for the standard curve was 0.9974, which is a very good relationship. In Table 2, the distance travelled by each band was measured and plotted on the table. This table shows how many bands are in each fish sample, how far they travelled, and if any other fish sample bands travelled the same distance. Table 3 organizes the information on Table 2 and shows the number of bands each fish sample has along with the number of bands they share in common with each fish sample. Figure 6 shows the final cladogram based off of experimental data. The constructed cladogram shows swordfish and yellow fin tuna sharing the most common ancestor rather than the yellow fin tuna and tilapia. In Figure 7, the results of the western blotting on the nitrocellulose membrane are shown on the left. The results could not be determined if they were accurate because actin and myosin were added to each sample, thus every band travelled the same distance. On the right side of Figure 7, the standard curve was constructed by plotting the distance travelled (x) against the molecular weight (y). From the standard curve, the correlation coefficient was 0.98, which is great. In Table 4, the molecular weight of each fish sample was compared to the distance travelled.

Conclusion: The data obtained in the experiment was very similar to the hypothesis formed. Initially, it was believed that the tilapia and the yellow fin tuna would have the most common ancestor, however, it was the swordfish and the yellow fish tuna that had the most common ancestor, sharing 4 protein bands. During the gel analysis, there was blobs of proteins together; this could be partly due to pipetting errors and too much of each fish sample being added due to higher concentrations. The western blot was supposed to show the myosin light chain, a protein that is essential for muscles. When western blotting, error could have been reduced by ensuring there were no bubbles during the sandwich blotting. This can be done by using the roller after placing each layer on the blotting sandwich. In our experiment, the addition of actin and myosin altered our data collected, thus a proper evaluation could not be made. However, because actin and myosin are such essential proteins, they are in each fish sample. The data could have been properly collected if the control was not added to each of our samples. There was also a few pipetting errors that could have caused lighter bands to appear when there should not have been some. If the experiment were to be further continued, the fish samples could be compared to other types of fish to determine if those fish are more closely related. Using western blotting, different types of proteins can be tagged aside from actin and myosin to see if less common proteins are shared amongst different species. By doing so, the whole experiment can be related back to central dogma. The similar proteins in each fish can be traced back to the RNA, which comes from the DNA, which leads to common ancestors. This whole process can be taken even further to show evolutionary changes over a specific length of time.

Works Cited: 1) Lab Manual 2) FishBase...