Event Title

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.

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Apr 11th, 10:00 AM Apr 11th, 11:30 AM

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.
http://cornerstone.lib.mnsu.edu/urs/2017/poster-session-A/26