title: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Neurobiology, University of Pittsburgh, 200 South Lothrop Street, Pittsburgh, PA 15213, USA; Departments of Neurobiology and Neurology, St. Joseph's Hospital and Medical Center and Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, USA. Electronic address: mac230@pitt.edu.
authors.forename: Mahlon A
authors.initials: MA
authors.lastname: Collins
authors.affiliation: Departments of Neurobiology and Neurology, St. Joseph's Hospital and Medical Center and Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, USA. Electronic address: Jiyan.An@dignityhealth.org.
authors.forename: Jiyan
authors.initials: J
authors.lastname: An
authors.affiliation: Departments of Neurobiology and Neurology, St. Joseph's Hospital and Medical Center and Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, USA. Electronic address: Danielle.Peller@dignityhealth.org.
authors.forename: Danielle
authors.initials: D
authors.lastname: Peller
authors.affiliation: Departments of Neurobiology and Neurology, St. Joseph's Hospital and Medical Center and Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, USA. Electronic address: Robert.Bowser@dignityhealth.org.
authors.forename: Robert
authors.initials: R
authors.lastname: Bowser
mesh_terms: D000418:Albumins / Q000378:metabolism; D015153:Blotting, Western; D002556:Cerebrospinal Fluid Proteins / Q000032:analysis* / Q000378:metabolism*; D004396:Coloring Agents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D016014:Linear Models; D008297:Male; D013129:Spinal Puncture; D013194:Staining and Labeling; D013334:Students; D014495:Universities
chemical_list: D000418:Albumins; D002556:Cerebrospinal Fluid Proteins; D004396:Coloring Agents; D049452:Green Fluorescent Proteins
keywords: Cerebrospinal fluid; Loading control; Total protein stain; Western blot