Capillary electrophoresis of insulin-like growth factors: Enhanced ultraviolet detection using dynamically coated capillaries and on-line solid- phase extraction

Matthew E. Roche, Marlys A. Anderson, Robert P. Oda, Lawrence B. Riggs, Michael A. Strausbauch, Ryo Okazaki, Peter J. Wettstein, James P. Landers

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Insulin-like growth factor-I and -II (IGF-I and IGF-II) are difficult to separate and measure as a result of their homology, beth structurally and immunologically. A number of binding proteins (BPs) which interact with the IGFs with high affinity complicate the ability to measure the IGFs accurately and reproducibly. Current methodology for measuring IGF is immuno-based and involves dissociation from the IGFs and removal of the binding proteins through sample acidification and removal by solid-phase adsorption. However, the net result is an assay that is time-consuming and, at best, semiquantitative. In an attempt to improve the reproducibility and accuracy of IGF-I and -II measurement, electrophoretic systems employing dynamically coated and bare silica capillaries were evaluated. Separations in bare silica capillaries in the presence or absence of the cationic additive, decamethonium bromide were ineffective for resolving IGF-I and IGF-II. However, when the capillary was coated dynamically with polybrene, IGF-I and -II could be resolved in a BSA sample matrix using a low pH buffer. Despite the fact that the IGFs could be resolved in the presence of an IGF-I analog used as an internal standard, polybrene recoating was required after as few as 12 runs and poor coating-to-coating reproducibility was observed. Use of polydiallyldimethylammonium chloride (PDMAC) as a dynamic cationic coating and a low pH buffer containing 0.5% PDMAC was found to be much more effective, providing reproducible separation of IGF-I and -II. It was found that PDMAC need not be included in the separation buffer to obtain reproducible analyses regarding IGF separation. Subsequently, functionality remained intact for as many as 35-40 consecutive analyses before recoating was required. Without the need for PDMAC in the buffer, on-line solid-phase extraction-capillary electrophoresis could be accomplished for detection of IGF-I and -II at concentrations as low 196 ng/mL.

Original languageEnglish (US)
Pages (from-to)87-95
Number of pages9
JournalAnalytical Biochemistry
Volume258
Issue number1
DOIs
StatePublished - Apr 10 1998

Fingerprint

Capillary electrophoresis
Solid Phase Extraction
Capillary Electrophoresis
Somatomedins
Insulin-Like Growth Factor I
Insulin-Like Growth Factor II
Chlorides
Buffers
Hexadimethrine Bromide
Silicon Dioxide
Coatings
Carrier Proteins
Insulin-Like Growth Factor Binding Proteins
Acidification
Adsorption
Assays

Keywords

  • Binding proteins
  • Capillary electrophoresis
  • Coated capillary
  • IGF-I
  • IGF-II
  • PDMAC
  • Polybrene
  • Solid-phase extraction-capillary electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Capillary electrophoresis of insulin-like growth factors : Enhanced ultraviolet detection using dynamically coated capillaries and on-line solid- phase extraction. / Roche, Matthew E.; Anderson, Marlys A.; Oda, Robert P.; Riggs, Lawrence B.; Strausbauch, Michael A.; Okazaki, Ryo; Wettstein, Peter J.; Landers, James P.

In: Analytical Biochemistry, Vol. 258, No. 1, 10.04.1998, p. 87-95.

Research output: Contribution to journalArticle

Roche, Matthew E. ; Anderson, Marlys A. ; Oda, Robert P. ; Riggs, Lawrence B. ; Strausbauch, Michael A. ; Okazaki, Ryo ; Wettstein, Peter J. ; Landers, James P. / Capillary electrophoresis of insulin-like growth factors : Enhanced ultraviolet detection using dynamically coated capillaries and on-line solid- phase extraction. In: Analytical Biochemistry. 1998 ; Vol. 258, No. 1. pp. 87-95.
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