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Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

After having established the basic protocol of two-dimensional electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt) in 1988 (A. Görg et al., Electrophoresis 1988, 9, 531-546), some critical parameters of the actual IPG-Dalt protocols as well as the results obtained with horizontal and vertical second-dimensional sodium dodecyl sulfate-electrophoresis are demonstrated and discussed.

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.

An interlaboratory comparison was conducted on the positional and quantitative reproducibility of yeast proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using isoelectric focusing with immobilized pH gradient (pH 4-7) in the first dimension. The basic experimental set-up was as follows: one laboratory prepared and distributed a [35S]methionine-labeled total yeast protein extract (Göteborg, Sweden), another laboratory prepared the IPG strips to be used by all labs in this study (Munich, Germany), the third laboratory (Aarhus, Denmark) circulated the protocols and coordinated the modest attempts to unify them. Samples were run horizontally in the first dimension and vertically in the second. The gels were sent to Göteborg for processing by phosphoimager technology and computerized image analysis (PDQuest), and the 2-D PAGE resolved proteins were located and quantified automatically. A subset of 470 spots was manually matched in all gels out of an average of 1328 resolved proteins. The positional interlaboratory comparison revealed great pattern reproducibility, the correlation coefficient in no case being less than 0.9994. In absolute terms an average deviation of 2.8 mm (x-position) and 1.8 mm (y-position) were obtained for all nine gels (three gels per lab). The interlaboratory comparison of protein quantitation displayed higher variability, and the best correlation coefficient generated was 0.975. An average standard deviation of 34.5% was calculated for protein quantitation including all three labs, a value slightly higher than the intralaboratory variation (range 20-28%). Thus, despite differences in protocols, chemicals and equipment, the immobilized pH gradient technology gave extremely high positional and quantitative reproducibility. This will greatly facilitate the exchange of data and the establishment of multi-user image-based 2-D gel databases.

An intra- and interlaboratory comparison of positional reproducibility of protein spots in two-dimensional electrophoresis using immobilised pH gradients (IPG) in the first dimension (IPG-DALT) was made. Aliquots of two different samples, human cardiac and barley leaf proteins, were separated in two different laboratories (London and Munich), using 180 mm long IPG gel strips, pH 4-8, for the first dimension and homogeneous SDS-PAGE gels (12% T) for the second dimension. Subsets of 340 (cardiac) and 200 (barley) well-resolved spots distributed across the 2-D gel patterns were selected for computer analysis (PDQUEST) of positional reproducibility. The IPG-dimension was highly reproducible in each laboratory, with a mean standard deviation of about 1 mm for both types of sample. Interlaboratory comparisons revealed identical results for barley with a mean standard deviation along the x-axis of about 1 mm, whereas the cardiac matchset showed slightly more variability (mean standard deviation approximately 1.5 mm). Nevertheless, IPG-DALT provides significantly improved reproducibility of spot positions compared to conventional isoelectric focusing with synthetic carrier ampholytes.

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