PubMed · 8220773
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
U Gerischer, P Dürre. 1993. Sequencing using custom designed oligonucleotides.. https://doi.org/10.1385/0-89603-248-5%3A75
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
A rapid, sensitive, semiautomated method to measure the activity of mast cell-derived tryptase has been developed. The assay is based on the tryptase-mediated hydrolysis of vasoactive intestinal peptide that was modified to include an N-terminal dansyl reporter group. Tryptase cleaves vasoactive intestinal peptide (VIP) producing two major and two minor products. Full-length VIP was separated from the proteolysis products by reverse-phase (C18) chromatography. The amount of each product as well as the amount of full-length substrate remaining was determined using an in-line fluorescence detector. As little as 0.5 pmol of peptide could be detected. Predominant cleavages were after Arg-14 and Lys-20 with minor cleavages after Lys-15 and Lys-21. The hydrolysis of VIP at Arg-14 was slightly affected by the presence of the dansyl group at the N-terminus. While the Km value was unaffected, the kcat value decreased, yielding a kcat/Km ratio that was eightfold lower. The addition of calcium chloride to the reaction mixture resulted in a slight decrease in the kcat/Km ratio. Cleavage of dansyl-VIP by tryptase was completely inhibited by DesPro2-Arg15-Aprotinin.
A fast method is reported for the precise and accurate quantification of cytokine mRNA, based on a quantitative polymerase chain reaction (PCR) assay. Post-PCR detection of the amplification products is achieved using an automated electrochemiluminescent (ECL) detection system. The target is amplified using a biotinylated forward and a tris(2,2'-bipyridine)ruthenium (II) (TBR)-labeled reverse primer. The amplification products are then captured on streptavidin coated paramagnetic beads and quantified by measuring the ECL signal of the TBR label. The results obtained are reproducible and accurate over a wide range (3 orders of magnitude) of concentrations. Quantitative results can be obtained using a standard curve which is generated with a synthetic external standard. This technique was applied to quantify tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin-2(IL-2) mRNA levels in human T cells transfected with the corresponding genes.
Explore the source record for details and available documents.