Quantitative Dot Blot Analysis(QDB)Reveals The Variation Of Housekeeping Proteins At Tissue And Cell Levels

Objective:Housekeeping proteins,which mainly function in cellular maintenance,are commonly used as endogenous controls as they are often considered to be adequately and stably expressed.However,an ideal protein for this purpose does not actually exist.Cells are exposed to rapidly changing microenvironments as a result of various changes in metabolic conditions.To survive these stresses,cells must adopt various strategies to increase their adaptability to the rapidly changing microenvironments.An inappropriate endogenous control often drastically affects the accuracy and reliability of the results and may even completely subvert the outcomes.Therefore,clarifying the stability of housekeeping proteins is very important for proteomics.In order to clarify the level of changes in housekeeping proteins in tissues and cells,we used quantitative dot blot analysis(QDB),a high-throughput research method for this study.Methods:(1)In this part,14 TRAMP mice(C57BL/6)were selected for the study.The contents of six housekeeping proteins in 14 mice liver tissues were detected by Western Blot and the levels of housekeeping proteins among different mice liver tissues were analyzed.(2)According to the anatomical structure,the livers of three TRAMP mice were divided into 10 parts.The levels of 6 housekeeping proteins in 10 different parts of the liver were detected by Western Blot analysis,and the levels of housekeeping proteins in different parts of the same tissues were analyzed.(3)The entire prostate of 87 mice(including 40 WT,47 TRAMP)was harvested in this study.Mice were divided into WT mice group and TRAMP mice group,and then stratified by week age(12W,16?18W,20?30W).Quantitative dot blot analysis(QDB)was used to detect the level of Tubulin in prostate tissues.The content and the stability of Tubulin were detected.(4)Eight human cell lines were selected and the content of Tubulin in the cells was determined by Western Blot and QDB analyses.The content of Tubulin was analyzed in different cells.(5)To construct HEK293T monoclonal cells(human renal epithelial cell line transfected with adenovirus E1A gene),in total 12 groups were cultured.Western Blot and QDB analyses were used to detect the contents of Tubulin in 12 groups of cells.To determine whether the amount of Tubulin in the same cell was consistently expressedResults:A comprehensive analysis of the expression of housekeeping proteins in tissues was unstable.The expression of 6 housekeeping proteins in liver tissues was detected by Western Blot analysis and the instability of expression was detected.The content of Tubulin in prostate tissues was further detected by QDB method.Wild mice or TRAMP mice in the same or different week of age,the expression levels of Tubulin were inconsistent and significant differences.In addition,we detected the instability of housekeeping proteins in cell experiments by Western Blot analysis and the accurate concentrations were not measurable.We further detected the absolute content of the housekeeping protein Tubulin by QDB analysis.It was cleared that the level of Tubulin in cells was relatively stable.Conclusions:The housekeeping proteins can present significant variation between individual subjects.Conclusions based on relative measurements are inherently limited.Our study raises the necessity of providing the absolute levels of housekeeping proteins in high-throughput studies to avoid biased explanation of the results at the protein level.