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

Abstract

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.

Comparative 2-DE Protein Analysis in a 3-D Geometry Gel

“…For processing of the 3-D image stack and data evaluation, we use ImageJ software from the National Institutes of Health (NIH;rsb.info.nih.gov/ij), as described in our previous work (4). To visualize the separation patterns (Figure 2C), vertical sections representing a series of 36 conventional 2-DE slab gels are generated out of the 3-D image stack using the plugin TransformJ (www.imagescience.org/meijering/software/transformj).…”

“…For casting, the gel cassette is removed from the instrument and sealed with a plate on the bottom. The gel solution is covered with a floating plate to prevent the access of ambient air during polymerization, as described previously (4). …”

“…Examples for one-parameter high-throughput separation are the quality validation of purified proteins prior to crystallization (4) and the analysis of high-performance liquid chromatography (HPLC)-prefractionated proteins by SDSPAGE. The loading in a 2-D array on top of the 3-D gel results in a very high sample capacity in which the density is limited only by lateral diffusion.…”

“…The loading in a 2-D array on top of the 3-D gel results in a very high sample capacity in which the density is limited only by lateral diffusion. For efficient loading, we described a method for simultaneous electrokinetic transfer of all samples from 96-, 384-, or 1536-well microtiter plates directly onto the 3-D gel (4). …”

The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter

“…They are based on small scale combinations of different variables like growth conditions, lysis buffers, type of constructs, host strains, fusion tags, co-expression of molecular chaperones and addition of osmolytes to identify promising solutions to be verified in large scale purification [ 1 - 8 ]. The resulting numerous samples can be screened using protocols based on SDS-PAGE or dot-blot to discriminate between promising and negative results [ 8 - 10 ].…”