Total protein is an effective loading control for cerebrospinal fluid western blots

Abstract

Background: Cerebrospinal fluid (CSF) has been used to identify biomarkers of neurological disease. CSF protein biomarkers identified by high-throughput methods, however, require further validation. While Western blotting (WB) is well-suited to this task, the lack of a validated loading control for CSF WB limits the method's accuracy.

New method: We investigated the use of total protein (TP) as a CSF WB loading control. Using iodine-based reversible membrane staining, we determined the linear range and consistency of the CSF TP signal. We then spiked green fluorescent protein (GFP) into CSF to create defined sample-to-sample differences in GFP levels that were measured by WB before and after TP loading correction. Levels of CSF complement C3 and cystatin C measured by WB with TP loading correction and ELISA in amyotrophic lateral sclerosis and healthy control CSF samples were then compared.

Results: CSF WB with the TP loading control accurately detected defined differences in GFP levels and corrected for simulated loading errors. Individual CSF sample Western blot and ELISA measurements of complement C3 and cystatin C were significantly correlated and the methods showed a comparable ability to detect between-groups differences.

Comparison with existing method: CSF TP staining has a greater linear dynamic range and sample-to-sample consistency than albumin, a commonly used CSF loading control. The method accurately corrects for simulated errors in loading and improves the sensitivity of CSF WB compared to using no loading control.

Conclusions: The TP staining loading control improves the sensitivity and accuracy of CSF WB results.

Keywords: Cerebrospinal fluid; Loading control; Total protein stain; Western blot.

Figure 1

CSF Total Protein (TP) Linearity. (A) Left, Representative Coomassie stained gel of a serial dilution of increasing amounts of CSF TP ranging from 0.25 μg to 40 μg from the same sample. Right, quantification of the TP signal. Square root transformed TP amount (x axis) and sum total normalized, square root transformed integrated density (y axis) are plotted in a line-connected XY scatter graph. Data are shown as the mean ± SD for triplicate experiments using separate pooled samples. (B) Same as (A), but for PVDF membrane stain. (C) Same as (A and B), but for albumin Western blot. (D) Comparison of the untransformed values obtained by each method over the range of 0.25-20 μg total protein loading. Data are shown as the mean ± SD for triplicate experiments using separate pooled samples.

Figure 2

CSF Total Protein Consistency. The consistency of the CSF total protein signal was evaluated by loading 5 μg of CSF total protein from 8 separate pooled samples and measuring the resultant integrated density. (A) Representative Coomassie stained gel. (B) Same as (A), but for PVDF membrane stain. (C) Same as (A and B), but for albumin WB. (D) Summary of results. The CV ([standard deviation/mean] × 100) of quadruplicate experiments was determined.

Figure 3

Corrective Performance of CSF Total Protein (TP) Loading Control. (A) Left, Representative images of in-tandem varying spiked GFP and CSF TP. Right, quantification of the corrected (red squares) and uncorrected (green triangles) normalized integrated density values obtained for each band. (B) Left, representative images of a serially diluted CSF TP stained membrane and equal loading GFP WB are shown. Right, quantification of the resultant uncorrected and corrected normalized integrated density values against the true value based on GFP concentration. (C) Left, representative images of a constant TP stained membrane and two-fold difference loaded GFP WB are shown. Right, the resultant fold-difference obtained from the uncorrected and corrected values is plotted against the true two-fold difference for triplicate experiments. Plots in (A) and (B) represent the mean ± SD of triplicate experiments.

Figure 4

Application of Total Protein (TP) Loading Correction to Candidate ALS CSF Biomarkers. (A) Representative TP stained membrane of 5 μg total loading for 5 healthy control and 5 ALS CSF samples. (B) Representative WB of complement C3 in the CSF of above healthy control and ALS CSF samples. (C) Plot comparing normalized, relative individual subject C3 levels obtained by ELISA and TP corrected WB from the above CSF samples. (D) Plot comparing normalized, relative between-groups differences for TP, C3α, C3β, total C3 WB, and total C3 ELISA. * = corrected p < 0.05. (E-H) Same as (A-D), respectively, but for cystatin C in a second set of 5 ALS and 5 healthy control CSF samples. Plots A-D represent the mean ± SD of triplicate WB experiments, E-H quadruplicate WB experiments, and all plots show quadruplicate ELISA results.