Characterization Protocol - Part 1 PDF

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Characterization Protocol - Part 1...

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Name Kelly Hewes MCB 253 - H12 15 February 2019 SDS-PAGE Characterization Lab Protocol Background: Protein electrophoresis is the widely used method to separate proteins based on their unique charged properties, using an artificial electric field for separation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a one of the protein electrophoresis methods used in order to determine the molecular weight of proteins. This method separates proteins by gel electrophoresis using a discontinuous polyacrylamide gel and sodium dodecyl sulfate (SDS). Native polyacrylamide gel electrophoresis (Native PAGE) is another viable method for protein characterization but this technique is better utilized to determine the aggregation state of a protein. SDS-PAGE denatures proteins and gives them a negative uniform charge which allows the proteins to be separated based on size alone, with the smaller polypeptide chain migrating further than the larger polypeptide chains. A Native PAGE separates whole proteins based on both the size, charge, and shape of macromolecules. Considering this information, the SDSPAGE method is best suited for determination of molecular weight of proteins and characterization of the unknown protein. Native PAGE will cause the molecular weight of the proteins to be influenced by the aggregation state and the charge of the proteins, while SDSPAGE will solely provide the molecular weight uninfluenced by other factors. During the experiment, we will be using the Sample Buffer + BME (10%) + SDS (12 mL sample buffer with SDS, 9.6 mL dH2O, 2.4 mL BME). BME stands for beta- mercaptoethanol or 2-mercaptoethanol. It is a thiol reducing agent that disrupts the intramolecular and intermolecular

disulfide bonds to achieve complete protein denaturation and maintain proteins in their fully reduced states (Bio-Rad, 2012). Since SDS-PAGE requires the protein to be denatured to its primary structure, BME is a good candidate in assisting the unfolding process of the protein and ensuring the success of the SDS-PAGE experiment. SDS is a detergent that causes the cell membrane to dissolve and coats the proteins with negative charges that destroy most of the protein’s complex, causing the resultant proteins to take on a uniform charge-to-mass ratio proportional to their molecular weights. This allows different proteins with similar molecular weights to migrant similarly. We will also be using a 4-20% gradient agarose gel (Bio-Rad) instead of the 10% or 12% discontinuous PAGE gel (Bio-Rad Mini-Protean TGX Precast Gel). It is distinguished that a higher percentage of agarose enhances resolution of lower molecular weight bands while a lower agarose percentage better separates larger bands (Yilmaz, Ozic, & Gok, 2012). Since the protein of interest is unidentified, the molecular weight is undistinguishable and a particular percentage of stacking gel is difficult to choose. Under these circumstances, a gradient gel would best to separate and distinguish the molecular weight of the unknown protein. We will be staining with Coomassie blue stain (Bio-Rad 161-0786) because this dye will allow the visualization of proteins as blue bands on a clear background. The other option was performing a western blot with ponceau s stain. Ponceau is able to stain membranes like the nitrocellulose membrane used in a western blot but gels are better stained with Coomassie blue stain. Coomassie blue stain will permeate the gel, stain the protein and fix the protein in place to aid in visualization. Coomassie blue is also nonhazardous and offers a short staining time, making it the preferred material to use for SDS-PAGE. Kaleidoscope protein weight marker (Bio-Rad 161-0324) is the standard Kaleidoscope protein ladder that will be utilized to provide a standard of comparison for the data acquired. Kaleidoscope protein

standard is a Precision Plus Protein standard that has a staining technology that provides batchto-batch molecular mass consistency and reproducible migration (Bio-Rad, 2012). We will also be using 10X electrode buffer with SDS (30.4 g tris base, 144.2 g glycine, 10 g SDS). This is a running buffer that will keep the pH of the gel at a specific level for application of the protein during electrophoresis. The tris base (30.2 g) is a buffer maintaining a pH range between 7.07 and 9.07. Glycine (144.2 g) is an amphoteric amino acid that will lost a hydrogen atom (H+) or accept a H+ depending on the pH and state of the environment. SDS, as mentioned above, is a detergent that prepares proteins for electrophoresis. Overall, SDS-PAGE is used to separate proteins based on molecular weight based on the migrating distance of bands on the electrophoresis gel. The purpose of this experiment is to determine the molecular weight (in Daltons) of the unknown protein and utilize this information to narrow the unknown protein to a possible 2-3 known proteins. The hypothesis is that SDS-PAGE will produce band migration lengths which can be used to identify the molecular weight of the unknown protein and aid in uncovering the identity of the unknown protein. Protocol: Create the following dilution samples of the unknown protein and the sample buffer solution in labeled test tubes. Test Tube Unknown Protein

Sample Buffer

1

3.3 uL of unknown protein #2 6.7 uL of the sample buffer and BME and SDS buffer

2

2 uL of unknown protein #2

8.0 uL of the sample buffer and BME and SDS buffer

3

0.67 uL of unknown protein #2

9.33 uL of the sample buffer and BME and SDS buffer

Within this dilution scheme, the 1 ug protein dilution generated 0.67 uL of unknown protein and since this value is below 2 uL (the minimum quantity a micropipette can pipette), this sample will not be created. Mix the solutions and heat the samples at 95 degrees Celsius for 5 minutes in a heat block. Obtain a precast 4-20% gradient agarose gel. Place the agarose gel into the casting tray, with the short plate facing inwards. Make sure the other side of the casting tray is balanced with another cast gel or a dummy plate. Secure the tray in place using the clamps on each side. Place the tray into the tank and add 10X electrode buffer with SDS to the inner chamber (continue to fill the box until the gel is completely submerged). Next, remove the comb out carefully from the agarose gels. Use a micropipette to carefully load the samples and the standard ladder into the wells of the gel. 1. Load the assigned amounts of each sample into the appropriate wells. a. Lane 1: 10 uL Kaleidoscope Protein Standard Ladder b. Lane 2: 10 uL of test tube 1 c. Lane 3: 10 uL of test tube 2 After the wells have been loaded, add more 10X electrode buffer with SDS to partially fill the outer chamber of the tank up to the 2 gels line. Then, place the lid onto the chamber, attach the power supply, and run the samples in the agarose gel inside the electrophoresis chamber for 50 minutes at 200V. After the time has concluded, turn off the power supply and disconnect the gel from the power supply. When the gel is done running, remove the gel and stain it with Bio-safe Coomassie dye. Wash the gel 3 times for 5 minutes each in 200 mL of ddH20. Remove all the water from the staining container and add 10 mL of Bio-safe Coomassie stain or enough to completely cover the gel. Gently shake on a rocker for 1 hour. Protein bands will be visible within 20 minutes and reach maximum intensity within an hour. Rinse the gels in 200 mL of

ddH2O on a rocker. After conclusion, proceed to obtaining an image of the gel and utilize this information to calculate the molecular weight of the protein using the standard curve. Data: Table 1. SDS-PAGE Molecular Weight Standard Distances Molecular Weight (kDa)

Distance migrated (mm)

10 15 20 25 37 50 75 100 150 250 Molecular Size (kDa) as a Function of Migration Distance(mm) 1.2

Molecular Weight (kDa)

1

0.8

0.6

0.4

0.2

0 0

0.2

0.4

0.6

0.8

1

1.2

Distanceof Migration (mm)

Figure 1. A standard curve is produced by plotting the molecular weight against the distance of migration of the standard. A semi-logarithmic graph will be extrapolated where y and x represent molecular weight in kilo-Daltons and distance of migration in millimeters respectively. Expected Results:

SDS-PAGE will produce multiple stained protein bands on the gel. After performing the procedure, we expect to take an image of our gel after staining and these protein bands will be present on this picture. The distance these bands traveled on the gel will be utilized to calculate the relative migration distance (Rf) of the protein standards and the unknown protein #2. The migration distance can be determined by dividing the migration distance of the protein by the migration distance of the dye front. A ruler (in cm) would be a proper tool to measure the migration distance of the protein and the standards. Based on the Rf values obtained from the bands in the standard, a logarithm graph of the Molecular Weight (kDa) vs Distance of Migration (mm) can be plotted. The distance of migration will be the calculated Rf values. A linear equation will be generated from this graph and this will help determine the molecular weight of the protein. In the y = mx + b equation, the x will represent the migration distance in mm and the y value will represent the molecular weight of the unknown protein. The molecular weight will allow the unknown protein #2 to be narrowed down to a possible 2-3 known proteins that can serve as the identity of the unknown protein.

Citations:

Bio-Rad Laboratories, Inc. (2012, May 29). A Guide to Polyacrylamide Gel Electrophoresis and Detection. Retrieved from http://www.biorad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf Yılmaz, M., Ozic, C., & Gok, İ. (2012, April 04). Principles of Nucleic Acid Separation by Agarose Gel Electrophoresis. Retrieved from https://www.intechopen.com/books/gelelectrophoresis-principles-and-basics/principles-of-nucleic-acid-separation-by-agarosegel-electrophoresis...