No abstract

1. Hepatic microsomal UDP-glucuronyltransferase (EC 2.4.1.17) derived from either weanling or adult rats exhibits three pH optima, at pH 5.4, 7.2 and 9.2, when o-aminophenol is the acceptor substrate, whereas p-nitrophenol is the acceptor substrate only on pH optimum is observed, at pH 5.4.2. Prior treatment of rats of either age with 3-methylcholanthrene results in a 2-3-fold increase in o-aminophenol conjugation at pH 5.4 and a 6-9-fold increase at pH 9.2. At pH 7.2, the induced enzyme is 2 to 3 times more active towards o-aminophenol than the control enzyme, but no pH optimum is demonstrable. 3. o-Aminophenol conjugation at pH 5.4 and 9.2 is inhibited competitively by both p-nitrophenol and p-nitrophenyl glucuronide, suggesting that the two phenolic aglycones share the same binding site. At pH 7.2, however, p-nitrophenyl glucuronide does not inhibit o-aminophenol conjugation, suggesting that the binding site at this pH is not shared by the two phenols. These data are consistent with the existence of more than one binding site for o-aminophenol on UDP-glucuronyltransferase.

Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive.

No abstract

45Reliable quantification of protein extracts from tissues can be a challenge e.g. due to 46 interference of the high fat content in tissues of the nervous system. Further problems like 47 under-or overerstimation of protein concentrations in protein quantification kits like the 48 bicinchoninic acid (BCA) assay can occur. In addition, common lysis buffers such as RIPA 49buffer are known to be unable to solubilize a large amount of proteins (~10-30%) leading to 50 unsatisfactory and unreliable experimental results with techniques such as immunoblotting. 51In this work, we have developed a Ponceau S staining based protein quantification assay. 52This assay is compatible with tissues or cells directly lysed in 2x SDS gel loading buffer, 53 containing bromophenolblue, leading to more complete protein extraction. Protein 54 concentrations of several samples can be determined in a fast and cost-effective manner and 55 subsequent experiments (e.g. Western blot) can be performed without loss of proteins. The 56 presented protein quantification method is highly reliable, fast and economical. Using this 57 method, it is possible to save between 2300 to 3200€ per 1000 lysates as compared to the 58 costs of a commercial BCA kit. 59 60 61 62 63 64 109 2.3 Lysate Preparation 110 Cell lysates were prepared by direct lysis of cells in 2x SDS LB on the plate using a cell 111 culture scraper and subsequent pulse-vortexing for 10 seconds. Sciatic nerves of either the 112 right or the left side from two different mice were pooled and snap frozen in liquid nitrogen 113 immediately after isolation. The harvested brain was divided into two pieces and lysates were Ponceau Dot Blot for Protein Quantification Helbing, Böhm et al. 131 S solution (0.1% Ponceau S in 5% acetic acid) was applied for one minute on loaded 132 membranes and equal distribution was ensured. Afterwards the membrane was briefly 133 washed with DI water until background staining was removed and membranes were placed 134 into a plastic foil and scanned with a Epson Perfection V750 Pro scanner using the 135 professional mode and the reflective document type in the scanning software. 136 2.6 Protein quantification with Fiji and Microsoft Excel 137 After creating a greyscale 8 bit image in the free, open-source Fiji software, the rectangle tool 138 and ROI manager were used to define the different dots as regions of interest. The rectangle 139was always left at the same size for all dots to avoid variation in the "area" variable of the Ponceau Dot Blot for Protein QuantificationHelbing, Böhm et al.

Preconditioning with LPS induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are CNS-resident immune cells that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, – using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins Stat1/2.

No abstract