Determination of Optimally Resolving Gel Concentration and Migration Time (Path) in Gel Electrophoresis

Aldroubi, Akram; Zakharov, Sergey F.; Chrambach, Andreas

doi:10.1006/abio.1995.9976

Cited by 10 publications

(10 citation statements)

References 2 publications

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“…the LiCor 4300 DNA Analysis System (Li-Cor Biosciences, Lincoln, NE), should be suitable for protein analysis as well with an adaptation of the gel cassette to short separation distances, and using appropriate protein labeling dyes matching the specific excitation laser wavelength. However, with DNA sequencers a sample recovery is not possible, in contrast to specifically developed instruments like the HPGE-1000 (LabIntelligence, Menlo Park, CA) [37][38][39] and the Pharmacia PhastSystem (GE Healthcare). On the other hand, the capacity of these instruments is limited to 8 samples per run for the HPGE-1000 and 12 samples per gel (2 gels per run) for the PhastSystem, and the latter instrument does not provide online detection capability.…”

Section: Discussionmentioning

confidence: 99%

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels

Ventzki¹,

Stegemann²,

Martínez³

et al. 2006

Electrophoresis

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) still remains the most reliable and comprehensive analytical method for the evaluation of protein extracts. However, conventional SDS-PAGE is time-consuming and, thus, unpractical if several tens or hundreds of samples must be examined. We show that SDS-PAGE protein analysis can be automated using slab gel DNA sequencers and compare the instrument's performance with conventional SDS-PAGE in terms of resolution, sensitivity and sample capacity. Labeled protein bands are detected online by laser-induced fluorescence (LIF) and the acquired signals are electronically stored for further processing, avoiding gel staining and scanning. Appropriate software allows immediate display of recorded data and convenient evaluation. The method provides a higher sensitivity and dynamic range than conventional Coomassie-stained gels and the resolution of proteins with different masses is independent of the polyacrylamide concentration. Internal markers can also be used for direct quantification and assignment of the molecular masses. Additionally, we present a novel electrophoresis instrument for the simultaneous separation and online LIF detection of all samples of a microtiterplate in parallel lanes in a 3-D geometry gel cylinder. The specific gel thermostatting concept prevents irregular sample migration (smiling) and improves the reproducibility and comparability of individual separation patterns. In combination with the expected large capacity of 384 or 1536 samples, this makes the instrument a valuable tool for high-throughput comparative screening applications.

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Section: Discussionmentioning

confidence: 99%

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels

Ventzki¹,

Stegemann²,

Martínez³

et al. 2006

Electrophoresis

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“…Note that in some previous applications of that procedure more than a single electroelution step was required to obtain a nearquantitative disappearance of the protein peak of interest from the pattern (e.g. [11]). In application to rGFP, a single electroelution step sufficed for near-disappearance of the peak from the pattern.…”

Section: Electroeluatementioning

confidence: 99%

“…[2,10,11,14]). However, the disappearance of the fluorescent band from the gel pattern does not, by itself, guarantee the arrival of the electroeluted protein in the electroeluate buffer since (i) the protein may be located in the 100 mL of gel plugging the elution cup; (ii) the protein after migration into the electroeluate buffer may be damaged and/or its fluorophore lost, unless that buffer is periodically renewed.…”

Section: Critical Electroelution Strategymentioning

confidence: 99%

Mass spectrometric analysis of the electroeluates of fluorescent proteins after preparative electrophoresis in the automated HPGE-1000 apparatus

et al. 1999

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“…The reasons for using the discontinuous Tris-Tricinate buffer system are specified in [12]. The buffer system is also the one that has been applied in the protein separations, using the HPGE apparatus, from its inception [1,11,[13][14][15][16][17][18][19], and in previous work on the transfer of proteins from gel electrophoretic bands into MS [4,6].…”

Section: Buffermentioning

confidence: 99%

Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate- proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester

2001

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A gel electrophoresis apparatus capable of scanning the migration path fluorometrically and of computer-directed electroelution of bands was applied to the mass spectrometric identification of sequentially electroeluted 5(6)-carboxyfluorescein-N-hydrosuccinimide ester (FLUOS)-labeled sodium dodedyl sulfate (SDS)-proteins. The masses of four electroeluted SDS-proteins under study determined by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry are changed by 1% due to their reaction with FLUOS in a 1:5 molar ratio of protein:label, allowing for the identification of the labeled intact proteins on the basis of mass. More importantly, the partial (10 or 50%) derivatization of proteins with FLUOS does not preclude their tryptic hydrolysis, and identification of the protein on the basis of the mass spectrometric analysis of its tryptic peptides. Potentially, the procedure allows for the automated mass spectrometric identification of SDS-proteins globally labeled with FLUOS and electrophoretically separated, without need for any gel sectioning.

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Determination of Optimally Resolving Gel Concentration and Migration Time (Path) in Gel Electrophoresis

Cited by 10 publications

References 2 publications

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels

Mass spectrometric analysis of the electroeluates of fluorescent proteins after preparative electrophoresis in the automated HPGE-1000 apparatus

Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate- proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester

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