Reproducibility and quality assurance of two-dimensional gel electrophoresis of serum specimens.
Currently we are using two different ISO-DALT two-dimensional gel electrophoresis systems, designated MC-Iso 1 and MC-Iso 2, for the analysis of serum and plasma samples. Here we report quality-assurance data for both of these systems. CV values for the slopes of the pH gradient (ISO dimension) are 5.6% of less; CV values for the slopes of the molecular-mass curves (log Mr vs relative mobility in the DALT dimension) are 3.4% or less. We examined the various steps of the analysis in detail for reproducibility and protein loss, using radiolabeled albumin, alpha 2-macroglobulin, and beta 2-microglobulin. Generally, in the first dimension, less protein enters the MC-Iso 2 gels (our routine system in which silver stain is used) than enters the MC-Iso 1 gels (our wide-range system for myeloma serum samples, in which the gel is stained with Coomassie Blue), on the average, 87% as much. The CV at this stage for both systems is 5--8%. During equilibration, considerable amounts of protein are lost (approximately 30% in 10 min) from the ISO gel, and the reproducibility is also decreased. Resolution in the DALT dimension has, in most cases, little or no effect on either recovery or reproducibility. Overall, for most proteins expected to appear in an ISO gel of a given pH range, approximately 50--60% of the starting material may be expected to reside in the sodium dodecyl sulfate slab gel, under our conditions. The two most important variables affecting recovery are the concentration of the NaOH (used as catholyte) and the pH of the starting sample. The overall CV for the process is between 8 and 12%.
D001798:Blood Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008259:Macroglobulins; D008970:Molecular Weight; D011786:Quality Control; D012016:Reference Values; D012709:Serum Albumin
D001798:Blood Proteins; D008259:Macroglobulins; D012709:Serum Albumin