title: Labeling effects on the isoelectric point of green fluorescent protein.
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.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Canada.
authors.forename: D P
authors.initials: DP
authors.lastname: Richards
authors.forename: C
authors.initials: C
authors.lastname: Stathakis
authors.forename: R
authors.initials: R
authors.lastname: Polakowski
authors.forename: H
authors.initials: H
authors.lastname: Ahmadzadeh
authors.forename: N J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry; D005663:Furans / Q000737:chemistry; D049452:Green Fluorescent Proteins; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D008164:Luminescent Proteins / Q000737:chemistry*; D011804:Quinolines / Q000737:chemistry; D037401:Scyphozoa / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D005663:Furans; D008164:Luminescent Proteins; D011804:Quinolines; C063756:3-(2-furoyl)quinoline-2-carbaldehyde; D049452:Green Fluorescent Proteins