Analysis of intracellular regulatory proteins by immunoaffinity capillary electrophoresis coupled with laser-induced fluorescence detection.
Measurement of intracellular regulatory proteins is of great importance in many areas of biomedical research. In this communication we describe an antibody-based capillary electrophoresis system equipped with an on-line laser-induced fluorescence detector capable of measuring intracellular proteins in cultures as low as 100 cells. The system demonstrated a high degree of precision and accuracy, being capable of detecting the fluorochrome-labeled analytes of interest at concentration of approximately 0.5 pg. We have used this instrument to study concentrations of the intracellular regulatory proteins STAT-1 and STAT-3, following stimulation of lymphocyte cultures with the inflammatory cytokine, IL-6. Using a combination of four antibodies specific for either STAT-1 or STAT-3 in both their nonphosphorylated and phosphorylated forms, we were able to demonstrate the differential expression of these proteins over time.
Phillips
Terry M
TM
Ultramicro Analytical Immunochemistry, Division of Bioengineering and Physical Sciences, National Institutes of Health, Bethesda, MD 20892, USA. phillipst@ors.od.nih.gov
Smith
Paul
P
D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007834:Lasers; D012015:Reference Standards; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D013050:Spectrometry, Fluorescence / Q000379:methods*; D015534:Trans-Activators / Q000032:analysis*
D004268:DNA-Binding Proteins; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015534:Trans-Activators