Citation

Musyaju S, Modi HR, Flerlage WJ, Scultetus AH, Shear DA, Pandya JD. Anal. Biochem. 2023; ePub(ePub): ePub.

Copyright

(Copyright © 2023, Elsevier Publishing)

DOI

10.1016/j.ab.2023.115301

PMID

37673410

Abstract

Owing to evidence that mitochondrial dysfunction plays a dominant role in the traumatic brain injury (TBI) pathophysiology, the Western blot (WB) based immunoblotting method is widely employed to identify changes in the mitochondrial protein expressions after neurotrauma. In WB method, the housekeeping proteins (HKPs) expression is routinely used as internal control for sample normalization. However, the traditionally employed HKPs can be susceptible to complex cascades of TBI pathogenesis, leading to their inconsistent expression. Remarkably, our data illustrated here that mitochondrial HKPs, including Voltage-dependent anion channels (VDAC), Complex-IV, Cytochrome C and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) yielded altered expressions following penetrating TBI (PTBI) as compared to Sham. Therefore, our goal was to identify more precise normalization procedure in WB. Adult male Sprague Dawley rats (N = 6 rats/group) were used to perform PTBI, and novel REVERT Total Protein (RTP) method was used to quantify mitochondrial protein load consistency between samples at 6 h and 24 h post-injury. Notably, the RTP method displayed superior protein normalization compared to HKPs method with higher sensitivity at both time-points between experimental groups. Our data favors application of RTP based normalization to accurately quantify protein expression where inconsistent HKPs may be evident in neuroscience research.

Language: en

Keywords

Traumatic brain injury; Housekeeping proteins; Loading control; Mitochondria; REVERT total protein staining; Western blot