Preliminary Study On β-actin As A Loading Control For Plasma-bases Western Blot Analysis Of Major Depressive Disorder Patients

BackgroundMajor depressive disorder is a common mental illness, as a syndromeit involves a significant and lasting sadness, the lifetime prevalence is up to16%, but the pathogenesis of depression is yet not well understood, and thelack of objective diagnostic indicators. Since the plasma is an easilyobtained and widely used sample, detecting the changes of specific plasmaprotein expression become more and more popular in the diagnosis ofclinical disease. Recently, differential proteomics technology has distinctedadvantage in finding disease-related biomarkers, exploring and uncoveringmany unknown but important protein. Therefore, it is expected that lookingfor depression-related protein can provide an objective marker for earlydiagnosis of depression by means of plasma differential proteomicstechnology.Although the plasma differential proteomics technology has moreadvantages than the traditional method in finding disease-related biomarkers, the method has shortcomings and limitations. The primaryproblem is that differential proteins which are found by plasma differentialproteomics technology need to be validated to ensure the reliability of theresults. Herein, Western blot is known as a commonly used method forvalidation. In western blot analysis, it is important to choose a suitableloading control. β-actin is an important cytoskeletal proteins in cells, andmainly distributed in the cytoplasm, Owing to its relatively high andconstant expression, it has been widely used in Western blot of cell culturesand tissue extracts. Hereby, whether β-actin presents in the plasma? If inthis case, how much of the plasma protein is β-actin? And also couldβ-actin be used in the plasma from major depressive disorder as loadingcontrol? These series of issues should be further investigated.ObjectiveThe purposes of present study was to detect whether β-actin presentsin the plasma and further assessed the suitability of β-actin as loadingcontrol for plasma from major depressive disorder patients.Methods1. Six subjects with first episode drug Major depression disorder andsix normal subjects were recruited. Blood was drawn from median cubitalvein,5ml samples were collected, plasma was separated and stored untilanalysis.2. Detecting the concentration of β-actin in the plasma of depressed and control individuals by means of ELISA.3. Evaluating the linearity of β-actin immunostaining and loadedprotein amount by Western blot.4. Compared the expression of β-actin between the major depressiondisorder group and healthy control group by Western blot.Results1. It exists β-actin in the plasma of control and depressed individualsand the ratio of plasma β-actin to total protein is9.4×10-8:1.2. The linear relationship between protein loading amount and β-actinexpression is fine. The mean R2value was0.974±0.012.3. The data revealed no statistically significant difference between thetwo groups (P>0.05). Moreover, the total coefficient of variation for β-actinexpression in the two groups was9.2±1.2%.Conclusion1. β-actin is presented in human plasma, but less in its content oftissues and cells.2. Preliminary studies have shown that β-actin is a suitable LC forplasma-based Western blot analysis of MDD patients.