Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data. Protocol Linearization of the Bradford Protein Calibration Graph:The Coomassie brilliant blue protein assay, commonly known as the Bradford assay 1 , is widely used because of its rapid and convenient protocol as well as its relative sensitivity. Unfortunately, there is a large degree of curvature over a broad range of protein concentrations (Fig. 1). Therefore, only a narrow range of relatively high protein concentrations, 2-10 mg/ml BSA, is used for the calibration graph, which then better fits linear regression (Fig. 1, green). However, the nonlinearity requires protein concentration of the unknown samples to fall within the limited range of the calibration graph in order to avoid a large error, and it also reduces the accuracy within the limited range. The nonlinearity presents a serious problem in particular when microgram amounts of protein are not available, and it often requires multiple dilutions of the unknown samples.As noted in the original Bradford paper, "the source of the nonlinearity is in the reagent itself since there is an overlap in the spectrum of the two different color forms of the dye." 1 . In fact, three forms of the Coomassie brilliant blue dye are in acid-base equilibrium at the usual acidic pH of the assay 2 . The red, blue, and green forms have absorbance maxima at 470, 590, and 650 nm, respectively (Fig. 2). The blue is the form that binds the protein, forming a complex that intensely absorbs light at 594 nm 3, 4 (Fig. 2). Bradford also noted that "the background value for the reagent is continually decreasing as more dye is bound to protein" 1 (Fig. 3). Therefore, we attempted to calculate the reduction of the 590 nm background as increasing protein quantities are added, by measuring the change of absorbance at 450 nm, where the protein-dye complex does not absorb. We found that the decreasing background partially, but not fully, accounts for the nonlinearity (Fig. 4).We then hypothesized that the decrease in free dye concentration produces another distortion of the l...

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SummaryTight regulation of the production of the key pro-inflammatory cytokine tumour necrosis factor-a (TNF-a) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a synthetic phospholipid, named hereafter phospho-ceramide analogue-1 (PCERA-1), was previously found to suppress lipopolysaccharide (LPS)-induced TNF-a blood levels. We therefore investigated the in vitro anti-inflammatory effects of PCERA-1. Here, we show that extracellular PCERA-1 potently suppresses production of the pro-inflammatory cytokine TNF-a in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Specificity is demonstrated by the inability of the phospholipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF-a suppression and IL-10 induction by PCERA-1 were observed in macrophages when activated by Toll-like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA-1 does not block activation of nuclear factor (NF)-jB and mitogen-activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the antiinflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF-a suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.

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With the success of the Human Genome Project, large-scale systemic projects became a reality that enabled rapid development of the systems biology field. Systems biology approaches to host–pathogen interactions have been instrumental in the discovery of some specifics of Gram-negative bacterial recognition, host signal transduction, and immune tolerance. However, further research, particularly using multi-omics approaches, is essential to untangle the genetic, immunologic, (post)­transcriptional, (post)­translational, and metabolic mechanisms underlying progression from infection to clearance of microbes. The key to understanding host–pathogen interactions lies in acquiring, analyzing, and modeling multimodal data obtained through integrative multi-omics experiments. In this article, we will discuss how multi-omics analyses are adding to our understanding of the molecular basis of host–pathogen interactions and systemic maladaptive immune response of the host to microbes and microbial products.