 Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans
Authors: Takuji Oka, Yukako Katafuchi, Kohsai Fukuda, Keisuke Ekino, Masatoshi Goto and Yoshiyuki Nomura
GfsA is a fungal O- galactofuranosyltransferase involved in the biosynthesis of O- glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant
protein of this enzyme from two heterologous host organisms. However, GfsA could not be
expressed as a recombinant protein in either Escherichia coli (E. coli) or Saccharomyces
cerevisiae (S. cerevisiae). Therefore, we decided to employ Aspergillus nidulans (A. nidulans) as
the host organism, and produced a strain that expressed 3x FLAG-tagged GfsA using
chromosomal tagging. To confirm its expression, a solubilized protein was prepared from the
tagged strain and analyzed with an anti-FLAG antibody. The strain that expressed 3x FLAGtagged
GfsA produced a functional protein with a mass of approximately 67 kDa. The method
described in this manuscript allows purification of the GfsA-3xFLAG protein as expressed in A.
nidulans cells.
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Western Analysis of Histone Modifications (Aspergillus nidulans)
Authors: Alexandra Soukup and Nancy P. Keller
Western blotting allows for the specific detection of proteins and/or modifications of proteins by an antibody of interest. This protocol utilizes a crude nuclei extraction protocol for Aspergillus nidulans to enrich for histones and other nuclear proteins prior to gel electrophoresis. Post translational modifications of histones may then be easily detected. After electrophoresis, the selected antibodies are used to detect and quantify levels of the modifications of interest.
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Immunofluorescence Analysis of Yeast Protein
Author: Yuehua Wei
Many important regulatory proteins such as transcription factors are regulated through subcellular localization. Protein localization can be examined by fusing a GFP tag. However, GFP is relatively big in size, and potentially may affect correct protein localization. Several small tags have been developed, such as myc, HA or Flag. By using immunostain and fluorescence microscopy as described in this protocol, one can easily probe the regulation of a selected yeast protein with the application of the aforementioned small tags.
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Determination of D-galactofuranose Content of Galactomannoproteins in Aspergillus nidulans
Authors: Takuji Oka, Yukako Katafuchi, Kohsai Fukuda, Keisuke Ekino, Masatoshi Goto and Yoshiyuki Nomura
Galactofuranose (Galf) is a component of several polysaccharides and glycoconjugates in certain species of filamentous fungi. Galf residues are frequently found in Aspergillus glycoproteins, including N-glycans and O-mannose glycans that modify many cell wall proteins and extracellular enzymes. It is known that furanoses, contained in oligosaccharides, are detected as pyranoses after hydrolysis, and that D-galactopyranose is not contained in the galactomannoproteins of Aspergillus spp. To determine the levels of D-galactofuranose in galactomannoproteins extracted from Aspergillus nidulans (A. nidulans), we measured the amount of D-galactopyranose production after galactomannoproteins hydrolysis. The method described in this manuscript allows determination of the D-galactofuranose content of galactomannoproteins in Aspergillus spp.
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Enzymatic Activity Assays in Yeast Cell Extracts
Authors: Melike Çaglayan and Samuel H. Wilson
Saccharomyces cerevisiae (S. cerevisiae) (commonly known as baker’s yeast) is a model organism that has a similar upstream base excision repair (BER) pathway for the repair of methylated bases as that in mammalian cells, and it is very easy to maintain in the laboratory environment. Here, we described a method to prepare cell extracts from yeast to investigate their enzymatic activities. This protocol is a quick and efficient way to make yeast cell extracts without using commercial kits.
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Isolating RNA from the Soil
Authors: Jacqueline M Chaparro and Jorge M Vivanco
Next generation sequencing has allowed for the analysis and ability to identify the microbial communities present in the environment. While DNA extraction from environments (such as soil) have provided a wealth of knowledge regarding microbial communities there are drawbacks that one encounters when using DNA as opposed to RNA. RNA allows for the determination of the identity of the microbes that are active and present at a particular time point and thus gives a clear picture of what these microbes are actually doing at a specific point in time and under a set of conditions. Extracting RNA from soil is challenging due to the inherent inhibitors present in the soil such as humic acids. Here we describe modifications to the MoBio RNA PowerSoilTM total RNA isolation kit to reproducively extract total RNA from the soil.
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Minimum Inhibitory Concentration (MIC) Assay for Antifungal Drugs
Authors: Jinglin L. Xie, Sheena D. Singh-Babak and Leah E. Cowen
The Minimum Inhibitory Concentration (MIC) Assay is widely used to measure the susceptibility of yeasts to antifungal agents. In serial two-fold dilutions, the lowest concentration of antifungal drug that is sufficient to inhibit fungal growth is the MIC. Typically, 50% inhibitory (MIC50) or 80% inhibitory (MIC80) values are reported. To facilitate visualization of antifungal susceptibility data, heat maps are generated whereby optical density values are represented quantitatively with colour.
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