Anton Posch

Stain-Free technology as a normalization tool in Western blot analysis

Western blots are used to specifically measure the relative quantities of proteins of interest in complex biological samples. Quantitative measurements can be subject to error due to process inconsistencies such as uneven protein transfer to the membrane. These non-sample-related variations need to be compensated for by an approach known as normalization. Two approaches to data normalization are commonly employed: housekeeping protein (HKP) normalization and total protein normalization (TPN). In this study, we evaluated the performance of Stain-Free technology as a novel TPN tool for Western blotting experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a representative of the HKP normalization strategy. The target protein (TP) used for this study was MCM7, a DNA licensing replication factor, which was shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell lines (LCLs). We studied the regulation of MCM7 with a multiplex Western blotting approach based on fluorescently labeled secondary antibodies and found that Stain-Free technology appears to be more reliable, more robust, and more sensitive to small effects of protein regulation when compared with HKP normalization with GAPDH. Stain-Free technology offers the additional advantages of providing checkpoints throughout the Western blotting process by allowing rapid visualization of gel separation and protein transfer.

MicroPrep: Chip-based dielectrophoretic purification of mitochondria

We have developed a microfluidic system – microPrep – for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long‐term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging. Hence, microPrep yields tens of micrograms of enriched and purified mitochondria within hours. Western blots of mitochondria fractions showed that contaminating endoplasmatic reticulum was reduced by a factor 6 when compared with samples prepared by state of the art centrifugation.

Fractionation of complex protein mixtures by liquid-phase isoelectric focusing.

Protein fractionation is essential to uncovering low-abundance proteins in complex protein mixtures. Many common methods and techniques are used to fractionate proteins, including chromatography (size exclusion, affinity, ion exchange, etc.), electrophoresis, and solution chemistry. Regardless of the method employed, the ultimate goal of protein fractionation is to enable more protein analysis by todays current proteomics technologies, such as one- (1-DGE) or two-dimensional gel electrophoresis (2-DGE) and liquid-chromatography and tandem mass spectrometry (LC-MS/MS).The MicroRotofor isoelectric focusing (IEF) cell fractionates proteins in free solution according to their isoelectric point (pI). We demonstrate the ability of the MicroRotofor to enrich low-abundance proteins in mouse brain tissue, thus enabling further identification of potential biomarker candidates.

Use of proteomics in radiobiological research: current state of the art

Biological systems are complex, variable and to a great extent adaptive to environmental and occupational challenge such as ionising radiation, making the mathematical modelling of their behaviour a diYcult task. The required models need to be based on useful experimental data describing global eVects on a cellular, tissue and organ level. High-throughput technologies such as proteomics have been shown to be powerful tools on many areas of modern biology. In radiation biology, especially facing the question of possible adverse health eVects following exposures to low doses of ionising and non-ionising radiation, new and sensitive approaches are slowly gaining support. Epidemiological studies suggest that doses of ionising radiation much lower than previously assumed may cause adverse eVects on human health. However, the epidemio

Sample preparation guidelines for two-dimensional electrophoresis

Abstract Sample preparation is one of the key technologies for successful two-dimensional electrophoresis (2DE). Due to the great diversity of protein sample types and sources, no single sample preparation method works with all proteins; for any sample the optimum procedure must be determined empirically. This review is meant to provide a broad overview of the most important principles in sample preparation in order to avoid a multitude of possible pitfalls. Sample preparation protocols from the expert in the field were screened and evaluated. On the basis of these protocols and my own comprehensive practical experience important guidelines are given in this review. The presented guidelines will facilitate straightforward protocol development for researchers new to gel-based proteomics. In addition the available choices are rationalized in order to successfully prepare a protein sample for 2DE separations. The strategies described here are not limited to 2DE and can also be applied to other protein separation techniques.

2D-ToGo workflow: increasing feasibility and reproducibility of 2-dimensional gel electrophoresis

Abstract Two-dimensional gel electrophoresis (2-DE) is one of the most powerful methods for studying global protein profiles. However, due to the multiple manual steps involved in gel based processing it is challenging to achieve the necessary overall reproducibility for a reliable comparative analysis, especially between different laboratories. To improve the 2-DE technique for quantitative analyses we have set up a robust 2-DE workflow, called 2D-ToGo, which utilizes latest innovations concerning instrumentation, consumables and protocols. Quantitative data analyses indicate the high reproducibility between replicate gels processed at a single site (intra-laboratory variation: CV 20%). The data-sets of the inter-laboratory comparison revealed similar results displaying a variation of CV 23%. The technical improvements given by our 2-DE workflow have a positive impact on process robustness and most importantly, reproducibility. Accordingly, many of the well-known challenges for resolving and quantitating up to thousands of different protein components in a given biological sample are minimized.