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| Contact |
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Kalman Franka |
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| Title |
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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 |
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Miriam S. Arrell, Franka Kalman |
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| References |
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Electrophoresis 37 (2016) 2913–2921 |
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| Url |
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http://dx.doi.org/10.1002/elps.201600246 |
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| Abstract |
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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 proteinmust 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. |
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