Denaturing Protein Gels
Kaytlin Goodwin
Cedarville University
Introduction Electrophoresis experiments are conducted on proteins for the purpose of separating proteins based on their characteristics. The characteristics that can be examined during the experiment are dependent on the preparation of both the proteins and the gel medium through which they will run. Native gel electrophoresis experiments highlight and examine multiple protein characteristics, such as size, charge, and shape. This is possible because native proteins “…are in their normal, biologically active forms…” (EDVOTEK, 2009, p. 4). Another type of electrophoresis, known as “SDS gel electrophoresis” involves the denaturation of protein for the purpose …show more content…
First, we ignored pages 9 and 10 of the instructions (which describe the procedure for preparing the gel), because the 3% agarose gels containing SDS had already been prepared for us by Dr. Kuruvilla’s lab assistant. Once we obtained our gels in the lab, we followed Dr. Kuruvilla’s outline of the instructions listed on pages 11 and 13. We followed the preparatory instructions numbered 2-6 on page 11 exactly, but we modified the procedure prescribed on page 13. We used pre-stained proteins, as instructed on page 13, but we did not warm the samples prior to adding them to the wells. The proteins we used were bovine serum albumin (BSA), which we loaded in the first well on the left (see Figure 1); ovalbumin, which we injected into the second well; cytochrome c, which we added to the third well; and lysozyme, which we inserted into the fourth well from the left. The other two wells remained empty. After loading 20 μL of each protein sample into its respective well (as directed in the handout), we ran the electrophoresis portion of the experiment. We followed the general procedure described in the ‘Running the Gel’ section of page 13 of the handout, except that we let the proteins run for 30 minutes at 150 volts, instead of at 125 volts. After 30 minutes, we removed the gels, placed them in plastic containers, and set the containers on the Belly Dancer. Due to some problems with the …show more content…
Kuruvilla informed us that the BSA and ovalbumin proteins were both considerably larger than the cytochrome c and lysozyme. Applying this knowledge to our hypothesis, we believed that because the BSA and ovalbumin were heavier in terms of molecular weight, they would not travel as far towards the cathode as the cytochrome c and lysozyme would. Our experiment confirmed our hypothesis, as can be seen in Figure 1. The ovalbumin did move slightly closer to the cathode than the BSA did, indicating that ovalbumin has a slightly lower molecular weight. Overall, though, in comparison to the cytochrome c and lysozyme, the BSA and ovalbumin were both substantially heavier. The lighter proteins moved almost the exact same distance, implying that they have similar molecular weights. Because both cyctochrome c (Koppenol, Rush, Mills, & Margoliash, 1991, p. 546) and lysozyme (Cirillo, Spizzirri, Iemma, & John Wiley & Sons, 2015, sec. 5.5 para. 1) are both positively charged, it would be interesting to see how these proteins moved relative to each other in a native gel electrophoresis, in which shape would be introduced as a
Kaytlin Goodwin
Cedarville University
Introduction Electrophoresis experiments are conducted on proteins for the purpose of separating proteins based on their characteristics. The characteristics that can be examined during the experiment are dependent on the preparation of both the proteins and the gel medium through which they will run. Native gel electrophoresis experiments highlight and examine multiple protein characteristics, such as size, charge, and shape. This is possible because native proteins “…are in their normal, biologically active forms…” (EDVOTEK, 2009, p. 4). Another type of electrophoresis, known as “SDS gel electrophoresis” involves the denaturation of protein for the purpose …show more content…
First, we ignored pages 9 and 10 of the instructions (which describe the procedure for preparing the gel), because the 3% agarose gels containing SDS had already been prepared for us by Dr. Kuruvilla’s lab assistant. Once we obtained our gels in the lab, we followed Dr. Kuruvilla’s outline of the instructions listed on pages 11 and 13. We followed the preparatory instructions numbered 2-6 on page 11 exactly, but we modified the procedure prescribed on page 13. We used pre-stained proteins, as instructed on page 13, but we did not warm the samples prior to adding them to the wells. The proteins we used were bovine serum albumin (BSA), which we loaded in the first well on the left (see Figure 1); ovalbumin, which we injected into the second well; cytochrome c, which we added to the third well; and lysozyme, which we inserted into the fourth well from the left. The other two wells remained empty. After loading 20 μL of each protein sample into its respective well (as directed in the handout), we ran the electrophoresis portion of the experiment. We followed the general procedure described in the ‘Running the Gel’ section of page 13 of the handout, except that we let the proteins run for 30 minutes at 150 volts, instead of at 125 volts. After 30 minutes, we removed the gels, placed them in plastic containers, and set the containers on the Belly Dancer. Due to some problems with the …show more content…
Kuruvilla informed us that the BSA and ovalbumin proteins were both considerably larger than the cytochrome c and lysozyme. Applying this knowledge to our hypothesis, we believed that because the BSA and ovalbumin were heavier in terms of molecular weight, they would not travel as far towards the cathode as the cytochrome c and lysozyme would. Our experiment confirmed our hypothesis, as can be seen in Figure 1. The ovalbumin did move slightly closer to the cathode than the BSA did, indicating that ovalbumin has a slightly lower molecular weight. Overall, though, in comparison to the cytochrome c and lysozyme, the BSA and ovalbumin were both substantially heavier. The lighter proteins moved almost the exact same distance, implying that they have similar molecular weights. Because both cyctochrome c (Koppenol, Rush, Mills, & Margoliash, 1991, p. 546) and lysozyme (Cirillo, Spizzirri, Iemma, & John Wiley & Sons, 2015, sec. 5.5 para. 1) are both positively charged, it would be interesting to see how these proteins moved relative to each other in a native gel electrophoresis, in which shape would be introduced as a