Using Total Protein Stain as a Loading Control for Western Blot Analysis of SOD2
Location
CSU Ballroom
Start Date
11-4-2017 10:00 AM
End Date
11-4-2017 11:30 AM
Student's Major
Chemistry and Geology
Student's College
Science, Engineering and Technology
Mentor's Name
Theresa Salerno
Mentor's Department
Chemistry and Geology
Mentor's College
Science, Engineering and Technology
Description
A deficiency in the antioxidant enzyme Superoxide Dismutase (SOD), more specifically the SOD2 isoform, can lead to an increase in oxidative stress resulting from hyperglycemia. Most of this previous work has been focused on total SOD enzymatic activity, not specific isoform expression, and most of the studies have used diabetic models rather than dietary studies. In this study, rats were fed diets supplemented with sucrose and two other sweeteners, Stevia and saccharin. SOD2 expression was measured at the protein level using the Western blot technique. The initial objective of this project was to establish a proper normalization for SOD2 relative quantitation using the Western blot technique and an IR labeled secondary antibody. The Revert Total Protein Stain has been tested as a loading control with a Sigma Prestige antibody for SOD2. By comparing different protein levels on the blot, we have established a linear range for the detection of both total protein and the SOD 2 protein target and have optimized the technique as a successful quantitation tool for SOD2 protein relative expression. The technique will now be applied to measure SOD2 protein expression in the control and experimental kidney samples. This will first involve homogenization and extraction with a RIPA buffer followed by centrifugation and the quantitation by the BCA assay so that all protein samples are analyzed in the linear range of detection for the target and total protein.
Using Total Protein Stain as a Loading Control for Western Blot Analysis of SOD2
CSU Ballroom
A deficiency in the antioxidant enzyme Superoxide Dismutase (SOD), more specifically the SOD2 isoform, can lead to an increase in oxidative stress resulting from hyperglycemia. Most of this previous work has been focused on total SOD enzymatic activity, not specific isoform expression, and most of the studies have used diabetic models rather than dietary studies. In this study, rats were fed diets supplemented with sucrose and two other sweeteners, Stevia and saccharin. SOD2 expression was measured at the protein level using the Western blot technique. The initial objective of this project was to establish a proper normalization for SOD2 relative quantitation using the Western blot technique and an IR labeled secondary antibody. The Revert Total Protein Stain has been tested as a loading control with a Sigma Prestige antibody for SOD2. By comparing different protein levels on the blot, we have established a linear range for the detection of both total protein and the SOD 2 protein target and have optimized the technique as a successful quantitation tool for SOD2 protein relative expression. The technique will now be applied to measure SOD2 protein expression in the control and experimental kidney samples. This will first involve homogenization and extraction with a RIPA buffer followed by centrifugation and the quantitation by the BCA assay so that all protein samples are analyzed in the linear range of detection for the target and total protein.
Recommended Citation
Bean, Nicole. "Using Total Protein Stain as a Loading Control for Western Blot Analysis of SOD2." Undergraduate Research Symposium, Mankato, MN, April 11, 2017.
https://cornerstone.lib.mnsu.edu/urs/2017/poster-session-A/26
