Equal loading of GST or GST fusion proteins was confirmed with Coomassie blue staining. The agarose then was resuspended in 30 l of sample buffer separated by SDS-PAGE followed by immunoblot using the rabbit polyclonal anti-NuMA antibody with 1:2000 dilution. From this, 500 l of supernatant was added to 50 l of GST, GST-4.1N-N-terminal domain (NTD), or GST-4.1N CTD agarose, incubated with slow rotation for 1 hr, and washed three times with 500 l of lysis buffer each time. The plasmid Dexpramipexole dihydrochloride was isolated from a colony displaying -gal activity using glass beads, transformed into bacteria by electroporation, and then DNA sequenced. Positive clones were further evaluated for -gal expression by nitrocellulose filter lift assay as described (Walensky et al., 1998a). A total of 2 106 independent clones were screened, and positive interactive proteins were identified by selecting for His+ growth phenotype.

The transformation of the hippocampal and cortex cDNA library into yeast expressing the GAL4C4.1N CTD fusion was performed essentially as described (Walensky et al., 1998a). The 4.1N CTD plasmid was transformed into yeast using the lithium acetate-polyethylene glycol method (Ausubel et al., 1990). This was used to screen a rat hippocampal and cortex cDNA library cloned into pPC86 (Li and Snyder, 1995 Lai et al., 1998), containing the GAL4 transcription domain. The Dexpramipexole dihydrochloride C-terminal domain of 4.1N (4.1N CTD) was cloned into yeast expression vector pPC97 (containing the GAL4 DNA-binding domain) as a bait. Two-hybrid screening was conducted using Y190 yeast strain containing the HIS3 and -galactosidase (-gal) reporter genes and the pPC97 and pPC86 expression vectors. All chemicals not included above were purchased from Sigma (St. Vectorshield mounting UV-DDB2 medium was from Vector Laboratories (Burlingame, CA). Glutathione-Sepharose 4B was supplied by Amersham Pharmacia Biotech (Uppsala, Sweden). ?Leu DO supplement, ?Leu/?Trp/?His DO supplement, minimal SD agar base, and minimal SD base for yeast two-hybrid screen were from Clontech (Cambridge, UK). Plasmids were purified using a Maxiprep kit from Qiagen (Hilden, Germany). Protein concentration was determined by the Bradford method. Mouse monoclonal T7.Tag antibody was from Novagen (Madison, WI). Mouse monoclonal anti-cyclin D1, cyclin B1, and proliferating cell number antigen (PCNA) were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Protein A-conjugated agarose beads was also from Calbiochem. Mouse monoclonal anti-NuMA, anti-hemagglutinin (HA), anti-Myc, and rabbit polyclonal anti-HA antibodies were supplied by Calbiochem (La Jolla, CA). 5-bromo-4-chloro-3-indolylвЂ“d-galactopyranoside, and isopropyl-1-thioвЂ“d-galactopyranoside were purchased from Boehringer Mannheim (Indianapolis, IN). Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.п»їThis was used to screen a rat hippocampal and cortex cDNA library cloned into pPC86 (Li and Snyder, 1995 Lai et al., 1998), containing the GAL4 transcription domain. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates.

In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. This new approach is easy to perform and adaptable in most laboratories. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis.

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