Total Protein Staining is Superior to Classical or Tissue-Specific Protein Staining for Standardization of Protein Biomarkers in Heterogeneous Tissue Samples

Abstract

Protein detection techniques such as western blotting and ELISA rely on housekeeping proteins as standards for sample normalization. However, clinical or animal tissue specimens are heterogeneous due to presence of contaminating cell types and tissues (e.g., blood vessels and muscle) or cellular decay during tissue storage and isolation which may compromise protein integrity. This biological heterogeneity may invalidate the assumption that housekeeping proteins are invariable across various specimens. This study provides data that advocate for protein standardization based on total protein staining in rabbit posterior capsular tissues. We compared the classical normalization markers glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin (TUBB) with other proteins that have low variation in expression (i.e., FTL, FTH1, EEF1A1, TPT1) based on RNAseq data for human posterior capsular tissues. Histological examination revealed a high degree of qualitative variation in microscopic images of capsular tissue specimens. This variation is reflected by significant differences in specific protein signals for all housekeeping proteins as detected by western blot analysis. However, total protein staining, which combines the intensity of multiple gel electrophoretic bands, normalizes natural biological variation observed for individual housekeeping proteins and permits assessment of protein integrity. Therefore, we propose that normalization based on total protein staining increases accuracy of protein quantification of heterogeneous tissue specimen samples.

Keywords: housekeeping; protein quantification; tissue samples; total protein.

Figure 1.

H&E staining of rabbit posterior capsule specimens qualitatively illustrates the high degree of histological variation in tissue samples.

Figure 2.

Selection of novel protein normalization markers based on RNA-seq data. The data are derived from mRNAs (in reads per million mapped reads, RPKMs) derived from human posterior capsular tissues obtained from patients undergoing primary knee arthroplasty (A). Description of all primary (B) and secondary (C) antibodies utilized.

Figure 3.

Western blot analysis (A) and quantification (B) of housekeeping proteins.

Figure 4.

Staining of electrophoretically separated proteins (A), quantitation of total protein staining (B) and protein integrity scores (C). Integrity scores were calculated based on the total protein volume above 50kDa divided by the total protein below 50kDa. This value was then divided by the highest score of all samples and then multiplied by 100% to yield a percent protein integrity based off of the least degraded sample.