Dynamic labeling during capillary or microchip electrophoresis for laser-induced fluorescence detection of protein-SDS complexes without pre- or postcolumn labeling.
The analysis of proteins under denaturing conditions is routinely performed with SDS-polyacrylamide gel electrophoresis. The automated capabilities of CE, use of nongel sieving matrixes, and on-line optical detection by either ultraviolet (UV) absorption or laser-induced fluorescence (LF) promise to revolutionize this method. While direct on-line detection of proteins is possible as a result of their intrinsic ability to absorb light in the UV part of the spectrum (detection sensitivity comparable to Coomassie Blue staining of gels), LIF provides more powerful detection but requires pre- or postcolumn fluorescence labeling of the proteins. The development of a protocol analogous to that used for double-stranded DNA analysis, where fluorescent intercalating dyes are simply included in the separation medium, would simplify size-based protein analysis immensely. This would avoid the complications associated with covalent modification of the proteins but still exploit the sensitivity of LIF detection. We demonstrate that this is possible with CE and microchip detection by incorporating, into the run buffer, a fluorescent dye that interacts hydrophobically with protein-SDS complexes. Key to this is a dye that fluoresces significantly when bound to protein-SDS complexes but not when bound to SDS micelles. Comparison of electropherograms from CE-based denaturing protein analysis with UV and LIF detection indicates that the presence of the fluor does not alter separation of the proteins. Moreover, comparison with electropherograms generated from microchip electrophoresis with LIF detection shows that equivalent patterns can be obtained. Despite the unoptimized nature of this separation system, a dynamic labeling protocol that allows for LIF detection for proteins is attractive and has the potential to circumvent the tedious labeling steps typically required.
Jin
L J
LJ
Department of Chemistry, University of Virginia, Charlottesville 22901, USA.
Giordano
B C
BC
Landers
J P
JP
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D011506:Proteins; D012967:Sodium Dodecyl Sulfate