http://www.researchonline.mq.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:24313 Chicken IgY responses against mixtures of six high abundance human plasma proteins were studied across a range of abundances from 1:1 for all six proteins to where one protein predominated above the other five by ≤1000 fold. The ability of any protein in that mixture to mount an IgY response varied. In the 1:1 mixture, human IgG produced the highest and α1antitrypsin the lowest individual responses. However, increasing relative abundance of any protein over others increased the total IgY response (i.e., sum of all responses to each antigen) above those obtained for 1:1 ratios. Increasing relative abundance of any protein over others in the mixture (e.g., 1:10, 1:100 and 1:1000) resulted in variations in response to both the overexpressed antigen and to the other five "constant" proteins. The overriding trends were that as individual proteins became relatively more abundant, they elicited higher IgY responses, while the remaining proteins elicited constant or even decreased responses. This study demonstrates that the ability to mount an IgY response to complex plasma protein antigens (e.g., human plasma) is conclusively driven by a combination of individual antigen immunogenicity and the relative abundance of components within any mixture. 2013-02-27T05:27:36.570Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:23838 We report a highly sensitive method for rapid identification and quantification of rare-event cells carrying low-abundance surface biomarkers. The method applies lanthanide bioprobes and time-gated detection to effectively eliminate both nontarget organisms and background noise and utilizes the europium containing nanoparticles to further amplify the signal strength by a factor of ∼20. Of interest is that these nanoparticles did not correspondingly enhance the intensity of nonspecific binding. Thus, the dramatically improved signal-to-background ratio enables the low-expression surface antigens on single cells to be quantified. Furthermore, we applied an orthogonal scanning automated microscopy (OSAM) technique to rapidly process a large population of target-only cells on microscopy slides, leading to quantitative statistical data with high certainty. Thus, the techniques together resolved nearly all false-negative events from the interfering crowd including many false-positive events. 2013-01-24T03:21:19.025Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:22185 The hypersecreting mutant Trichoderma reesei RUT-C30 (ATCC 56765) is one of the most widely used strains of filamentous fungi for the production of cellulolytic enzymes and recombinant proteins, and for academic research. The strain was obtained after three rounds of random mutagenesis of the wild-type QM6a in a screening program focused on high cellulase production and catabolite derepression. Whereas RUT-C30 achieves outstanding levels of protein secretion and high cellulolytic activity in comparison to the wild-type QM6a, recombinant protein production has been less successful. Here, we bring together and discuss the results from biochemical-, microscopic-, genomic-, transcriptomic-, glycomic- and proteomic-based research on the RUTC30 strain published over the last 30 years. 2012-10-23T00:33:13.138Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:21029 This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation. 2012-08-24T16:10:37.997Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:21030 The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography- electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO a nd IgG, described in the second protocol of this series (10.1038/nprot.2012. 063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides. 2012-08-24T16:10:33.126Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:19848 Don't freeze! A native chemical ligation-desulfurization strategy has been employed for the convergent synthesis of a library of defined antifreeze glycopeptides and glycoproteins (AFGPs) ranging in size from 1.2 to 19.5 kDa (see picture). These AFGPs possessed the secondary structure of a polyproline type II helix and exhibited significant ice recrystallization inhibition and thermal hysteresis activity. 2012-06-14T08:00:30.681Z ]]>