In vitro translation reactions of HA-tagged vectors were performed using the TNT T7 Coupled Wheat Germ Extract System (Prom-ega). Reactions were diluted in TNE buffer (50 mM Tris pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% NP-40) and incubated overnight with streptavidin-coated beads (ThermoFisher) conjugated to 0.2 μM biotin-c-di-GMP (Biolog B098), c-di-AMP (Biolog B106), or cGAMP (Biolog C197). Pull-down products were washed three times in TNE buffer supplemented to 1 M NaCl and analyzed by SDS-PAGE and western blotting for HA. Purified protein products (0.5 pmol) were incubated with 100 pmol biotin-c-di-GMP in the presence or absence of 100 pmol non-biotin labeled c-di-GMP competitors in buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1% NP-40 for 2 h at 4°C. Streptavidin-coated agarose beads were then added and incubated for an additional 2 h. Beads were washed and protein lysate was analyzed by SDS-PAGE.
Biotin c di gmp
Manufactured by Biolog
Sourced in Germany
Biotin-c-di-GMP is a chemical compound used in laboratory research and assay development. It consists of a biotin moiety covalently linked to the bacterial signaling molecule cyclic di-GMP (c-di-GMP). This compound can be utilized as a tool in various biochemical and cell-based applications, enabling the detection, quantification, or manipulation of c-di-GMP-related processes.
Automatically generated - may contain errors
Lab products found in correlation
C di amp, by Biolog (1 mentions)
Cgamp, by Biolog (1 mentions)
Tnt t7 coupled wheat germ extract system, by Promega (1 mentions)
Streptavidin coated beads, by Thermo Fisher Scientific (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Irdye 800cw streptavidin, by LI COR (1 mentions)
Biotin cgmp, by Biolog (1 mentions)
Hl 2000 hybridization incubator, by Analytik Jena (1 mentions)
2 protocols using biotin c di gmp
1
In vitro pull-down assay of c-di-GMP/AMP/GMP
2
Quantitative Analysis of c-di-GMP Binding Interactions
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Ultraviolet (UV) cross-linking experiment with c-di-GMP were performed as described54 (link). Biotin-c-di-GMP, Biotin-c-di-AMP and Biotin-c-GMP were purchased from BioLog Life Science Institute, Bremen, Germany. Ethr (1 mg/mL) was incubated with 0, 1, 5, and 25 μM Biotin-c-di-GMP in PBS buffer at RT for 1 h, followed by exposure to 254 nm UV light with 120,000 μJ from HL-2000 Hybridization Incubator (UVP, California, US) on ice for 30 mins. Meanwhile, Ethr was also co-incubated with 25 μM Biotin-c-di-AMP and 25 μM Biotin-c-GMP, reacting as described above. The proteins were electrophorized on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk at RT for 1 h. After a slight washing, the membrane was probed with 1:5000 IRDye 800CW streptavidin (LI-COR Bioscience, Lincoln, Nebraska) at RT for 1 h, washed with TBST for 3 times. The results were recorded by a LI-COR Odyssey scanner (LI-COR Bioscience) and quantified by application software v3.0.21 of Odyssey infrared imaging system.
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