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Fluorescence imaging detection for capillary isoelectric focusing. 1995

X Z Wu, and J Wu, and J Pawliszyn
Department of Chemistry, University of Waterloo, Ontario, Canada.

A simple laser-induced fluorescence (LIF) imaging detector and an ultrasensitive LIF imaging detector are described for capillary isoelectric focusing (CIEF). An argon ion laser beam of 496.5 nm is used as excitation source. In the simple LIF imaging detector, the excitation beam is directed into a capillary column by an optic fiber array. In the ultrasensitive LIF imaging detector, the laser beam is first expanded, then is focused into the 4.5 cm long capillary column by a cylindrical lens. Fluorescence emission is detected by a charge-coupled device (CCD) camera. The feasibility and performance of the LIF imaging detector system for CIEF are first verified with a naturally fluorescent protein, b-phycoerythrin. Then, the ultrasensitive LIF imaging system is used as a detector for CIEF of proteins labeled with fluorescein isothiocyanate (FITC). Three FITC-labeled proteins (i) alpha-D-galatosylated FITC-albumin, (ii) insulin-FITC, and (iii) casein-FITC, are used as model samples. Fluorescence images of the model samples are measured during the CIEF process. The focusing of the protein samples is complete in about 1.5 min. The ultrasensitive detector's detection limits for the FITC-labeled proteins are at the level of 10(-10) M, and the mass detection limits are about 4.5 x 10(-17) mole, even though only 10% of the fluorescence emission is collected. Therefore, the method is capable of separating and detecting 10(-11) M or amole (10(-18) mole) level protein samples with a band-pass filter more specific to the fluorescence light.(ABSTRACT TRUNCATED AT 250 WORDS)

UI MeSH Term Description Entries
D007328 Insulin A 51-amino acid pancreatic hormone that plays a major role in the regulation of glucose metabolism, directly by suppressing endogenous glucose production (GLYCOGENOLYSIS; GLUCONEOGENESIS) and indirectly by suppressing GLUCAGON secretion and LIPOLYSIS. Native insulin is a globular protein comprised of a zinc-coordinated hexamer. Each insulin monomer containing two chains, A (21 residues) and B (30 residues), linked by two disulfide bonds. Insulin is used as a drug to control insulin-dependent diabetes mellitus (DIABETES MELLITUS, TYPE 1). Iletin,Insulin A Chain,Insulin B Chain,Insulin, Regular,Novolin,Sodium Insulin,Soluble Insulin,Chain, Insulin B,Insulin, Sodium,Insulin, Soluble,Regular Insulin
D007525 Isoelectric Focusing Electrophoresis in which a pH gradient is established in a gel medium and proteins migrate until they reach the site (or focus) at which the pH is equal to their isoelectric point. Electrofocusing,Focusing, Isoelectric
D007834 Lasers An optical source that emits photons in a coherent beam. Light Amplification by Stimulated Emission of Radiation (LASER) is brought about using devices that transform light of varying frequencies into a single intense, nearly nondivergent beam of monochromatic radiation. Lasers operate in the infrared, visible, ultraviolet, or X-ray regions of the spectrum. Masers,Continuous Wave Lasers,Pulsed Lasers,Q-Switched Lasers,Continuous Wave Laser,Laser,Laser, Continuous Wave,Laser, Pulsed,Laser, Q-Switched,Lasers, Continuous Wave,Lasers, Pulsed,Lasers, Q-Switched,Maser,Pulsed Laser,Q Switched Lasers,Q-Switched Laser
D010799 Phycoerythrin The metal-free red phycobilin pigment in a conjugated chromoprotein of red algae. It functions as a light-absorbing substance together with chlorophylls.
D002364 Caseins A mixture of related phosphoproteins occurring in milk and cheese. The group is characterized as one of the most nutritive milk proteins, containing all of the common amino acids and rich in the essential ones. alpha-Casein,gamma-Casein,AD beta-Casein,Acetylated, Dephosphorylated beta-Casein,Casein,Casein A,K-Casein,Sodium Caseinate,alpha(S1)-Casein,alpha(S1)-Casein A,alpha(S1)-Casein B,alpha(S1)-Casein C,alpha(S2)-Casein,alpha-Caseins,beta-Casein,beta-Caseins,epsilon-Casein,gamma-Caseins,kappa-Casein,kappa-Caseins,AD beta Casein,Caseinate, Sodium,K Casein,alpha Casein,alpha Caseins,beta Casein,beta Caseins,beta-Casein Acetylated, Dephosphorylated,beta-Casein, AD,epsilon Casein,gamma Casein,gamma Caseins,kappa Casein,kappa Caseins
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D012710 Serum Albumin, Bovine Serum albumin from cows, commonly used in in vitro biological studies. (From Stedman, 25th ed) Fetal Bovine Serum,Fetal Calf Serum,Albumin Bovine,Bovine Albumin,Bovine Serum Albumin,Albumin, Bovine,Albumin, Bovine Serum,Bovine Serum, Fetal,Bovine, Albumin,Calf Serum, Fetal,Serum, Fetal Bovine,Serum, Fetal Calf
D013050 Spectrometry, Fluorescence Measurement of the intensity and quality of fluorescence. Fluorescence Spectrophotometry,Fluorescence Spectroscopy,Spectrofluorometry,Fluorescence Spectrometry,Spectrophotometry, Fluorescence,Spectroscopy, Fluorescence
D016650 Fluorescein-5-isothiocyanate Fluorescent probe capable of being conjugated to tissue and proteins. It is used as a label in fluorescent antibody staining procedures as well as protein- and amino acid-binding techniques. FITC,5-Isothiocyanatofluorescein,Fluorescein (5 or 6)-Isothiocyanate,Fluorescein-5-isothiocyanate Hydrochloride,5 Isothiocyanatofluorescein,Fluorescein 5 isothiocyanate,Fluorescein 5 isothiocyanate Hydrochloride,Hydrochloride, Fluorescein-5-isothiocyanate

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