Estimation of protein concentration at high sensitivity using SDS-capillary gel electrophoresis-laser induced fluorescence detection with 3-(2-furoyl)quinoline-2-carboxaldehyde protein labeling
| Autor: | Franka Kálmán, Miriam Arrell |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Clinical Biochemistry Sensitivity and Specificity 01 natural sciences Biochemistry Analytical Chemistry Silver stain 03 medical and health sciences chemistry.chemical_compound Capillary electrophoresis Mole Furans Derivatization Laser-induced fluorescence Fluorescent Dyes Chromatography 010401 analytical chemistry Quinoline Electrophoresis Capillary Proteins Reproducibility of Results Sodium Dodecyl Sulfate Spectrofluorometer Fluorescence 0104 chemical sciences 030104 developmental biology chemistry Linear Models Quinolines Electrophoresis Polyacrylamide Gel |
| Zdroj: | ELECTROPHORESIS. 37:2913-2921 |
| ISSN: | 0173-0835 |
| Popis: | 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) is a sensitive fluorogenic dye, used for derivatization of proteins for SDS-CGE with LIF detection (SDS-CGE-LIF) at silver staining sensitivity (ng/mL). FQ labels proteins at primary amines, found at lysines and N-termini, which vary in number and accessibility for different proteins. This work investigates the accuracy of estimation of protein concentration with SDS-CGE-LIF in real biological samples, where a different protein must be used as a standard. Sixteen purified proteins varying in molecular weight, structure, and sequence were labeled with FQ at constant mass concentration applying a commonly used procedure for SDS-CGE-LIF. The fluorescence of these proteins was measured using a spectrofluorometer and found to vary with a RSD of 36%. This compares favorably with other less sensitive methods for estimation of protein concentration such as SDS-CGE-UV and SDS-PAGE-Coomassie and is vastly superior to the equivalently sensitive silver stain. Investigation into the number of labels bound with UHPLC-ESI-QTOF-MS revealed large variations in the labeling efficiency (percentage of labels to the number of labeling sites given by the sequence) for different proteins (from 3 to 30%). This explains the observation that fluorescence per mole of protein was not proportional to the number of lysines in the sequence. |
| Databáze: | OpenAIRE |
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