Abstract
Purpose :
Glaucoma carries a huge disease burden and understanding it is critical. Quantitative Western Blot (WB) analysis requires comparison of the target protein normalized to an internal control. However, the housekeeping proteins can be susceptible to oxidative stress, mechanical changes, and aging in the retina. Normalization to total protein was recently reported. We compared B-actin and total protein as internal controls in mice with increasing age and with elevated intraocular pressure (IOP).
Methods :
Mouse tissues were acquired following protocols that were ethically reviewed and approved by the Vanderbilt University Medical Center Institutional Animal Care and Use Committee, and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. We divided the eyes of 10-month old DBA/2J mice into two groups based upon: 1) normal IOP (NIOP; (<10mmHg) and 2) High IOP (HIOP; >10mmHg) measured by tonometer (TONOLAB, Icare). A third group consisted of eyes from 10-week DBA/2J mice. Samples consisted of three retinas. 7 WBs comparing relative quantities among the 3 groups using B-actin, and 7 WBs using total protein levels (REVERT Total Protein Stain, LICOR Biosciences, Lincoln) were run. Comparison of expression among the 3 groups was performed with one-way ANOVA (SigmaPlot 12.5, Systat).
Results :
A significant difference in B-actin levels was present among the 3 groups (P<0.001) with a significant increase in the HIOP group compared to the other 2 groups (Bonferroni Multiple Comparison, HIOP vs NLIOP P=0.006; HIOP vs 10-week old P<0.001). No significant difference in total protein staining was present among the 3 groups (P=0.274).
Conclusions :
Total protein staining may be the superior method for normalization of WB proteins in the mouse retina. There was an increase in B-actin levels in the DBA/2J HIOP retinas compared to B-actin levels in the other groups, suggesting an influence of IOP. Whereas, there was no significant difference in total protein staining among the 3 groups.
This is a 2021 ARVO Annual Meeting abstract.