Labeling effects on the isoelectric point of green fluorescent protein.
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.
Richards
D P
DP
Department of Chemistry, University of Alberta, Edmonton, Canada.
Stathakis
C
C
Polakowski
R
R
Ahmadzadeh
H
H
Dovichi
N J
NJ
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
D005456:Fluorescent Dyes; D005663:Furans; D008164:Luminescent Proteins; D011804:Quinolines; C063756:3-(2-furoyl)quinoline-2-carbaldehyde; D049452:Green Fluorescent Proteins