Improving precision in gel electrophoresis by stepwisely decreasing variance components

Schröder, Simone; Brandmüller, Asita; Deng, Xi; Ahmed, Aftab; Wätzig, Hermann

doi:10.1016/j.jpba.2009.04.034

Cited by 10 publications

(4 citation statements)

References 45 publications

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“…Meanwhile, another improved protein detection alternative is available which is based on signal detection in the near-infrared range. 38,39 Preliminary results using this sensitive detection method suggest a total error of just 5% RSD%. 38 Own works in longer series 39 seem to confirm these favorable findings for simple, one-dimensional separations.…”

Section: Discussionmentioning

confidence: 93%

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Quantitative Gel Electrophoresis: Sources of Variation

et al. 2008

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Gel electrophoresis is known for its often unsatisfactory precision. Percental relative standard deviations (RSD%) in a range of 15-70% have been reported. Therefore, an improvement of precision in quantitative 2-DE is necessary. In the present, study we have analyzed the work flow of 2-DE in detail to assess the main error sources. Potential major sources of variability for this technique include the transfer between first and second dimension, the analyst's expertise, and the staining or rather detection of separated proteins. The remarkable and completely irregular changes of the background signal from gel to gel were identified as one of the governing error sources. These background changes can be strongly reduced by the direct detection of the separated proteins using native fluorescence. More than a 3-fold better signal-to-noise ratio was found compared to Ruthenium-(II)-tris-(bathophenanthroline disulfonat) (RuBPS) and Coomassie staining, although the sample was used in an 800-fold lower concentration. This improvement together with well-defined peaks resulted in a better quantitative spot reproducibility of approximately 12-16% RSD%. Possibly, the variabilities due to detection and evaluation were already reduced to minor error components. However, according to the law of error propagation, the major error sources dominate the total error. To really prove the good detection and evaluation, these other sources of variability such as sample preparation, strip rehydration, protein loading, transfer between dimensions, interactions between gel and proteins, gel scanning, and spot integration have to be reduced next.

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Section: Discussionmentioning

confidence: 93%

“…38,39 Preliminary results using this sensitive detection method suggest a total error of just 5% RSD%. 38 Own works in longer series 39 seem to confirm these favorable findings for simple, one-dimensional separations.…”

Section: Discussionmentioning

confidence: 93%

Quantitative Gel Electrophoresis: Sources of Variation

et al. 2008

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“…Research efforts are being pursued to improve its precision and to develop efficient detection methods to reduce irregular changes in background signals. One of the challenges is to find relevant parameters having a significant impact on the quality of detection and identification of proteins and other analytes 30. The work reported in the present paper focused more on the separation capability of the newly designed PAAm/dextran gels compared to the commercial product Duracryl ™ .…”

Section: Resultsmentioning

confidence: 99%

Newly designed polyacrylamide/dextran gels for electrophoresis protein separation: synthesis and characterization

Ainseba-Chirani

Dembahri

Tokarski

et al. 2011

Polymer International

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Newly designed gels for electrophoresis protein separation were synthesized from acrylamide, N,N -methylenebis (acrylamide) and dextran mixtures. Radical polymerization was initiated by ammonium persulfate and N,N,N ,Ntetramethylethylenediamine. The time dependence of absorbance during polymerization was monitored using UV-visible spectroscopy. The exothermic polymerization process exhibited a sharp rise of temperature reminiscent of the Trommsdorff effect. The swelling kinetics of the synthesized gels was examined in deionized water and buffer solutions. One of the challenges was to find an alternative to commercial products, sold as mixtures with no detailed chemical contents, commonly used in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation. For this reason, a systematic comparison was made of the properties of one of the most commonly used commercial gels, Duracryl  from Genomics Solution Inc., and those of the synthesized polyacrylamide/dextran gels.

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“…When starting with a large number of parameters (factors) whose effect sizes are unknown, it is recommended to start with a screening design such as the Plackett-Burman design [133]. After the most influential parameters and their contributions to the total error are identified this way, their main effects, interaction, and statistical significance can be studied in more detail.…”

Section: Optimizing Assay Precision: Decreasing Variance Componentsmentioning

confidence: 99%

Data quality in drug discovery: the role of analytical performance in ligand binding assays

Wätzig

Oltmann-Norden

Steinicke

et al. 2015

J Comput Aided Mol Des

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Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development.

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Improving precision in gel electrophoresis by stepwisely decreasing variance components

Cited by 10 publications

References 45 publications

Quantitative Gel Electrophoresis: Sources of Variation

Quantitative Gel Electrophoresis: Sources of Variation

Newly designed polyacrylamide/dextran gels for electrophoresis protein separation: synthesis and characterization

Data quality in drug discovery: the role of analytical performance in ligand binding assays

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