Labeling effects on the isoelectric point of green fluorescent protein

J Chromatogr A. 1999 Aug 20;853(1-2):21-5. doi: 10.1016/s0021-9673(99)00687-1.

Abstract

We studied the effects of fluorescent labeling on the isoelectric points (pI values) of proteins using capillary isoelectric focusing with laser-induced fluorescence detection (cIEF-LIF). Specifically, we labeled green fluorescent protein (GFP) from the jellyfish Aequorea victoria with the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). cIEF-LIF was used to monitor the native fluorescence of GFP and showed pI changes in GFP's FQ-labeled products. Multiple labeling of GFP with FQ produced a series of products with pI values shifted towards a low pH. We verified cIEF-LIF results with traditional slab gel IEF. Our cIEF-LIF technique can routinely detect 10(-11) M of FQ-labeled protein, whereas traditional slab gel IEF with silver stain detection gives detection limits of 10(-7) M in the same samples.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Fluorescent Dyes / chemistry
  • Furans / chemistry
  • Green Fluorescent Proteins
  • Isoelectric Focusing / methods*
  • Isoelectric Point
  • Luminescent Proteins / chemistry*
  • Quinolines / chemistry
  • Scyphozoa / chemistry
  • Spectrometry, Fluorescence

Substances

  • Fluorescent Dyes
  • Furans
  • Luminescent Proteins
  • Quinolines
  • 3-(2-furoyl)quinoline-2-carbaldehyde
  • Green Fluorescent Proteins