Abstract The purpose of this experiment was to determine if column chromatography and gel electrophoresis separated Bovine Serum Albumin from Myoglobin while determining the size of the proteins. The hypothesis stated that cuvettes two through 5 will contain both BSA and Myoglobin. The hypothesis was tested using column chromatography and gel electrophoresis to measure which contained both proteins and which cuvette had a higher concentration of BSA and Myoglobin.IntroductionBSA or otherwise known as Bovine Serum Albumin is a protein which means that it is a macromolecule. Myoglobin is a protein as well.

BSA and Myoglobin are found in fractions two through four. Serum Albumin is found in blood and it stabilizes the extracellular fluid in the vertebrae( Shi et. al 2016). Myoglobin is found in cardiac and skeletal muscles of vertebrates when it binds to oxygen( Parray et. al 2017).

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Some possible qualities that could be used to separate proteins could be size and shape because if it is denatured in anyway then the whole function changes. The objective of this study was to determine if column chromatography separated BSA and Myoglobin when they were mixed together. The fractions that contained BSA would be fractions 2, 4, and 7 while the fractions that contained Myoglobin would be 1, 3, 5, 6. If you separate BSA and Myoglobin with column chromatography, stain with Bradford Assay, measure absorbance with spectrometer and compare absorbance to standard data then, fractions 7, 8, and 9 will contain both BSA and Myoglobin. MethodsTo begin the experiment the column was packed with gel beads that had been hydrated for a couple hours in a 1x PBS buffer.

It was then loaded into the column and sat overnight to settle in and pack together tightly which made the results have a more clear resolution. The tubes were labeled zero to nine on the lid. Now, 1-4 mL of buffer sat on top of the column bed. The yellow cap was removed and the buffer drained out until only 0.5 mL remained on the column and then the column was capped quickly afterwards. A sample of protein was acquired and a micropipette was used to add 1 mL of protein that consisted of both Myoglobin and BSA into the column making sure none of it was directly dispensed into the gel bed. After this was complete, the process of collecting the 10 samples was underway.

The yellow cap was removed and tubes were placed beneath the cap making sure that not a drop was missed. Once the tube had filled up to the 1 mL marker, the next tube was placed underneath the column and the lid was closed and the tube was then placed on ice. These steps were repeated until all ten tubes were filled up. The buffer was monitored and as soon as the mixed protein sample had entered the column bed completely and then 5 mL of elution buffer in 1 mL at a time and made sure that it ran down the sides until all 5 mL was added. When the column stopped dripping consistently, more elution buffer was added 1 mL at a time this was continued until all 10 microfuge tubes were filled exactly to the 1 mL line. Once the 10 fractions were collected the yellow cap was placed back on the column to stop the flow of any liquids. Fraction 0 was thrown away because it only contained the buffer and the remaining 9 were the tubes that were mixed with the sample.

Afterwards, a micropipette was used to transfer 50 ?l of each protein into cuvettes and they were then placed back on ice. 1250 ?l of Bradford Reagent was added to each of the cuvettes and they sat for 2 minutes so everything could mix with the protein. The fractions that turned blue were recorded and the higher the intensity of the blue, the higher the concentration of the protein was. The unlabeled cuvette was filled with  1250 ?L of Bradford Assay and placed inside of the spectrometer after being wiped off with a kimwipe.

This calibrated the spectrometer so that the spectrometer knew what 0 absorbance and 0 concentration was. Gel electrophoresis was used so that the protein concentration could be seen and to identify which fraction contained BSA and which fraction contained Myoglobin. Using a micropipette, portions were taken from each fraction and put into the wells in an agarose gel and an electric current forced the proteins to move across the gel. After the proteins had separated, Coomassie Brilliant Blue Staining was used to see both the intensity and location of the protein bands.

ResultsStandard data was found so that the relationship of concentration and absorbance could be found. As concentration increases, light absorbance increases as well. The equation found from the standard data was y=0.0808ln(x)-0.

0288. As concentration increases, light absorbance increases as well which made a positively correlated graph. (Figure 1). The Bradford’s Assay identified the proteins in fractions 2 through 6.

The concentration spiked in fractions two through four which made those three fractions the most concentrated. (Figure 2). From our results, it was inclusive since there were holes poked into the wells allowing the proteins to escape. The concentrations of each protein goes as follows: 0.7885 ?g/ 50 ?L, 2171.48 ?g/ 50?L, 1918.705 ?g/ 50 ?L, 2550.

53 ?g/ 50 ?L, 142.650 ?g/ 50 ?L, 15.565 ?g/ 50 ?L, 3.

43 ?g/ 50 ?L, 1.576 ?g/ 50 ?L, 0.7788 ?g/ 50 ?L.

(Figure 2). Out of the results, the gel electrophoresis shows that fractions two, three, and four all had the highest protein concentration because of the thick protein band. (Figure 3). The gel electrophoresis and the Bradford’s Assay showed similar results and they should have shown similar results because they serve the same purpose of identifying how concentrated a protein is.  According to the gel electrophoresis, fractions two and three contained BSA while seven and eight contained Myoglobin and four contained both BSA and Myoglobin. These are all shown in figure three where you can see which fractions contain which proteins and how much of each protein is contained in each protein. Discussion The results of this experiment were inconclusive since there were holes that had been poked into the bottom of the wells causing the proteins to run out of wells 4 through 6. However, fraction numbers 2,3,and 7 all contained proteins, two and three being BSA and 7 being Myoglobin.

It was hypothesized that fractions two through six would contain proteins but two through four would contain both proteins. This hypothesis cannot be accepted or rejected due to the fact that the data was inconclusive because of the holes being poked in the wells that caused all of the protein to escape from the wells (Figure 3). Both proteins were found in the middle fractions because both proteins are similar in size. BSA is 66 kDa and Myoglobin is 17 kDa which is where you can start to see the size similarity even though the numbers do not seem to be super close in similarity. The protein antigen Streptococcus mutans hold a 190 kDa protein(Lapirattanakul et. al 2015).

BSA was found earlier in the fractions because it is larger even if it is larger but just a little bit it is still a larger protein which means it flows faster and does not get caught up in everything that is trying to filter out all the toxins(Lapirattanakul et. al 2015). One of the errors that occurred in this lab was the holes that were poked into the wells causing all of the protein to escape from the wells which left our data inconclusive and unreadable. This could have been prevented by having a sturdier hand and previous experience with working with micropipettes. Another error that could have taken place could have been if when collecting the protein samples some of the protein was spilled which would give us a false reading or no reading at all because the whole lab was based on the concentration of the proteins. A new question could be how does the absorbance level change if you change the proteins that you are measuring but keep the concentrations the exact same? If the two proteins were changed but concentrations were left the same then, your absorbance would fluctuate between increase and decrease as your concentration increases. For this you would end up testing concentration to make sure they are all the same and then using the spectrometer afterwards to test for absorbance and see the relationship between the two.

Works CitedLapirattanakul J, Nomura R, Matsumoto-Nakano M, Srisatjaluk R, Ooshima T, Nakano K. 2015. Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans. Int. J. Med. Microbiol. 305(3): 383-391.

Savadkoohi S, Bannikova A, Kasapis S, Adhikari B. 2014. Structural behaviour condensed bovine serum albumin systems following application of high pressure. Food chem. 150: 469-476Shi J, Pan D, Wang X, Liu T, Jiang M, Wang Q.

2016. Characterizing the binding interaction         between antimalarial artemether (AMT) and bovine serum albumin (BSA): Spectroscopic and molecular docking methods. J.

Photochem. Photobiol. B, Biol.

162: 14-23.Parray Z, Shahid S, Ahmad F, Hassan I Md, Islam A. 2017. Characterization of intermediate state of myoglobin in the presence of PEG 10 under physiological conditions. 99: 241-248Gervais D, Downer A, King D, Kanda P, Foote N, Smith S.

2017. Robust quantitation of basic-protein higher-order aggregates using size-exclusion chromatography. J Pharm Biomed Anal.

139: 215-220.Figure1. As concentration increases so does absorbance which is why there is a consistent logarithmic increase between concentration and absorbance levels as read by the LabQuest. Figure2. The concentration increases sporadically however, our results are inconclusive due to the holes poked in the wells that caused the proteins to escape which is why 1, 6-9 all look the same because there  is little to no protein in there. Figure3.  There were holes poked into wells 4-6 causing all of the protein to escape from the gel which means the location of BSA and Myoglobin are not known and the results are inconclusive.