This relationship was graphed and provided logarithmic function which could be used to find the size of an unknown protein just by measuring the distance that it revealed and plugging it to the equation. This is an important technique because the separation of proteins can reveal which polypeptides form the protein and identifying it can provide information on its purpose for its functionality and further scientific research in all types of fields such as the medical field.
Introduction Proteins are a large biological molecules that are made up of polypeptide chains. These chains connect to one another, but they are composed of smaller subunits called up amino acids. There exists 20 distinct amino acids which can be UT together into sequences in the primary protein structure. In the secondary protein structure, the sequence of amino acids connect into a chain with the aid of hydrogen bonds and form repeating patterns of either beta sheet or alpha helix.
In the tertiary structure, the patterns of amino acid chains combine to make a 3-dimensional folding pattern and in the quaternary structure, these 3- dimensional chains of amino acids combine to form a protein. Proteins are good to serve different functions in our bodies. For starters, proteins are able up act as enzymes that speed up reactions in our body. Proteins are also capable of serving as antibodies to help protect our bodies from being invaded by outside substances or organisms.
They are also an important part of the nervous system because proteins receive and respond to molecular signals that come from inside or outside the organism. Proteins also serve as a boat that transports substances within the organism and they can also regulate how a gene will be interpreted. The protein serves these functions and many more. In the laboratory experiment, proteins are denatured so that it is possible to see the primary structure of the protein. In order to achieve this, the process called electrophoresis is applied along with SD PAGE.
The way it works is by applying SD (sodium decoded sulfate) to the protein and then placing the protein into the mini protean gel rig. The SD has a negative charge due to the sulfate’s negative charge so when it is applied to the protein, the SD provides an evenly distributed charge by mass to the protein. Then when the protein is placed into the well of the gel and then current is applied to the system this is the PAGE (polysaccharide gel electrophoresis) stage ” the polypeptides move room away from the negative charge towards to positive charge and the proteins will move in proportion to their size (Dulia 2005).
Since the proteins travel a distance that is proportional to their size, I predicted that the proteins with less mass will travel a longer distance than the proteins with more mass, which will travel a short distance. The proteins are moving because of the force which the electric field created by the current exerts of these negatively charge proteins.
If the same force is applied to all proteins, then using Newton’s second law of harmonicas , I can see that the proteins with a higher mass will have a lower acceleration, which means that there will be a smaller change in velocity, with then means that that there would be a smaller changer in distance traveled; the proteins with less mass will have a greater acceleration, which means that it will have a greater change in velocity, which then means that there would be a greater change in distance traveled. After proteins are done moving from their initial positions, the gel is taken out and then stained for photographing purposes.
In order to calibrate this experiment, a maker protein is used in order or other proteins to be compared to it. This allows for a more accurate judgment when it comes to finding what the unknown protein was. This laboratory activity allows students to interact with laboratory materials and enact the technique of how to separate proteins using SD PAGE. In the process, students should be able to obtain a better understanding of how the amino acids chains are held together by intermolecular forces since the procedure is about denaturing proteins.
After collecting the appropriate data, students should be capable of putting the data together and showing a graphical presentation of the data, which will result in a curve-like graph. Materials and Method Activity l: SD PAGE This laboratory experiment required the use the following materials: ice bucket containing unknown protein samples, sample of protein casein, and high molecular weight stander; gloves, dry bath of ICC, micro centrifuge, 20 pipettes, mini protean gel; ring, power supply, ready gels, box odd long pipette tips, used tips cup, and a timer.
Begin this experiment by obtaining the proteins samples from the ice bath and placing them into the dry bath of ICC for 5 minutes. After the time is up, place he protein samples back on the ice and do not remove them until needed. Then, using a pipette, take 10 of each protein sample and place them in the wells in the following order: casein in well 9, first unknown in well 8, marker in well 7, second unknown in well 6, and another casein n well 5. Run a IV current through the gel for 45 minutes but if IV cannot be applied, make a note of the voltage that is applied.
When it is time, remove the gel from the mini protean gel container and place it in plastic container on the orbital shaker with Accompanies Brilliant Blue stain. Leave the orbital shaker on for 40 minutes. Remove the gel and dispose of the blue stain in the “used stain jar,” wash with distilled water, and dispose of the water down the drain. Then disdain the gel using distilled methanol/acetic acid for 20 minutes on the orbital shaker and after time is up, remove the gel and let the TA image the gel and dispose of the disdain in the “used disdain jar”.
Before imaging, however, rinse the gel with distilled water. Print the images of the gel and clean the area where the experiment was performed. In the case of this laboratory activity, the results obtained may have been effected by a couple of deviations performed. The voltage applied was IV rather than IV which speeds up the migration but affect the distance traveled and may not be proportional to the size of the protein. The time that was the proteins were left to migrate under the current was changed to 50 minutes since it was left running an additional 5 minutes.
And finally, the staining portion time was decreased by 5 minutes so the total time that was left running was 35 minutes rather than 40 minutes. This may have affected the quality and precision of the image. Results The following graph illustrates the relationship between mass and distance traveled by a protein. The equation of the curve is a logarithmic functions which relates the mass and distance traveled by a protein. The RE value is also included to show the reliability of the x and y values relationship.
Below is a picture of the gel after electrophoresis and staining which shows bands of proteins that traveled a certain distance. The wells are labeled one through five and each well contains a certain protein. Wells 1 and five contain casein, wells 2 and 4 contain different unknowns and well 3 contains a marker which serves as a control for the experiment. The mass of the casein was calculated using the equation from the chart and plugging in the distance traveled by each protein 51 mm, mm, and mm. The masses result 28. Kodak, 25. KDE, and 17. 1 KDE so the total mass would be 71. Kodak. Determining the Size of the Unknown Polypeptides In order to determine the size of the unknown polypeptide, one will use two things: the distance traveled by the polypeptide and the equation from the Weight of Protein vs… Distance Traveled graph in which the variable “x” is the distance traveled and the ‘Y’ variable is the mass. For unknown in well 2, the distance traveled by the polypeptide was 37. Mm. When it is plugged in to the equation, the mass of unknown I is 65. Asked.
Using the list of possible unknowns, bovine serum album seems to be the closest match with only a 1. 7% difference in mass. For unknown J in well 4, the distance traveled by the polypeptides were 46. Mm and 50. Mm and when plugged into the equation, the masses come out to be 39. Kodak and 30. Kodak. Since the polypeptides come from the same protein, their masses can be combines to find the unknown protein, which then results to be 69. Kodak. Using the list of possible unknowns, bovine serum album seems to be the closest match with a 5. 59% mass difference.
Discussion The data recorded from the marker in well 3 was used to create a standard curve in excel which provided a logarithmic equation of . The x-variable in this equation is the distance traveled by the protein and the y-variable is the mass. If taken another look at the graph, it can be noticed that as the mass decreases, the greater the distance the protein travels but if a best fit line is drawn, this relationship between mass and distance do not express a linear function cause it leaves out a big gap for error and does not accurately take into account all of the points from the plot.
A power best fit line, however, is somewhat close to taking into account all the plot points but it still leaves room for error because the linear best fit line is only good towards the low polypeptide weight and even then, there would still be a large error gap between the resulting mass using the linear equation and the actual mass of the protein, When placing a power best fit line, the trending equation that comes into result is where x is the distance traveled and y is the mass.
In using this equation to find out the mass of the protein that traveled mm in well 3, the mass comes out to be asked but the actual mass of the same protein is asked, a 3. 1% which is not bad at all, but the power curve is only good at the middle and lower molecular weight and could make a difference when it comes to finding the mass of unknown proteins. The logarithmic best fit line, on the other hand, provides a nearly perfect trending that touches all points and provides a good representation of all the plots in the graph at both high, middle, and low polypeptide molecular weight.
After carefully choosing the best fit line for the graph, it is possible to calculate the masses of the unknown proteins and possibly identify. As previously stated in the last two paragraphs of the results question, the logarithmic curve can give the masses of the unknown proteins by plugging in the distance traveled by the protein for the x-variable. For unknown l, the distance traveled was 37. Mm and the resultant mass would be 65. Asked. In the case of unknown J, it is necessary to add the resultant masses of the two individual bands that traveled 46. Mm and 50. Mm. The masses equate to 39. Kodak and 30. KDE and when added together, the resultant mass was 69. Kodak. Both of the unknowns have a mass close to that of bovine serum album’s mass which is 66. Kodak. When analyzing casein, it can be seen that both casein from wells 1 and 5 have three bands that traveled nearly the exact same distance for the same amount of time, we can conclude that the experiment was performed well. Because there is three noticeable bands, it can be concluded that casein is composed of at least three polypeptides an making it multimedia protein.
It is very common to find millimeters proteins in organisms because millimeters proteins can perform arioso complex functions due to the protein’s role in genetics and everywhere else in our body. For instance, journal entry in the Nature (International Weekly Journal of Science) from the University of Cambridge Department of Chemistry and Laboratory of Molecular Biology provided information that states that if protein does not fold accordingly, it could lead to medical conditions which can be damaging to the human body such as Alchemist’s disease and diabetes (Wright 2005).
Diabetes has been proven to be liked to genes and is therefore hereditary and can be passed on to future generations (Davies 1994). Similarly, he improper folding of proteins amongst other organisms can cause negative side effects. If the millimeters proteins are unable to perform their functions due to improper folding, then this means that the organism has a chance of not being able to survive.
If this occurs in multiple organisms of the same species, then this can lead to a decrease in the population of the specie, which then leads to extinction. Proper folding and diversification of the proteins, however, can lead to a successful expansion of the specie because the multimedia proteins aid the specie’s survival. The diversification of multimedia proteins would be a pathway o the emergence of new adaptations that the cell which in turn would lead for a greater population of a specie (Lynch 2012).
Conclusion This experiment had three main objectives to it allow students to interact with laboratory materials and enact the technique of how to separate proteins using SD PAGE, obtain better understanding of how the amino acids chains are held together by intermolecular forces since the procedure is about denaturing proteins, and presenting a graphical representation of the data, which will result in a curve-like graph.
The first objective was accomplished while the procedure of he experiment was taking place and the second objective was accomplished after the experiment was performed and seeing that the proteins separated into bands and that each band traveled a certain distance. This also provided a relative relationship between the distance traveled and the mass of the protein which was graphed using excel.
The graph presented that the mass of the protein and distance traveled by the protein existed as logarithmic function and the logarithmic function was also used to find the masses of the unknown proteins using the distance traveled. This graph proved my hypothesis correct hat those proteins with small masses would travel further than those proteins with larger masses, which would travel smaller distance. There were factors, however, that may have affected our data.
There were supposed to be nine bands on the marker in well three but due to the resolution of the photograph, only seven bands were noticeable and only six bands were used to make a graph using excel. This may have affected the equation of the graph due to the exclusion of two data points that could have added precision to the graph but the equation obtained from the graph using only six data points was satisfactory cause the percent differences between the unknown proteins and possible protein (bovine serum album ) were . % and 5. 59%. The denaturing of proteins serves for many purposes in the scientific field. Separating the proteins into polypeptides helps scientists such a biologists and chemists study the composition of proteins and also study the individual polypeptides. One way that SD PAGE could be used is for genetic engineering. Because it is possible to separate the proteins into smaller subunits, then this is theoretically possible to replicate a protein, however, this is only easier said than one.