c-di-GMP binding assays were carried out as previously described (Freedman et al., 2009 (link)). In brief, recombinant PlzA and PlzB were spotted onto nitrocellulose membranes and air dried. Rrp1 served as a negative control. The membranes were incubated with [32P]c-di-GMP (2 nM) alone, or in combination with 750 nM of c-di-GMP, GTP, GMP, ATP, cGMP, or cAMP (Sigma; PBS with 1% non-fat dried milk; 2 h; room temperature), washed twice with PBS (10 min) wrapped in cellophane and exposed to film overnight with intensifying screens at −80°C.
C di gmp
Manufactured by Merck Group
Sourced in Germany, United States
C-di-GMP is a cyclic dinucleotide molecule that functions as a secondary messenger in various bacterial species. It plays a key role in regulating cellular processes such as biofilm formation, virulence, and motility. The core function of C-di-GMP is to transduce environmental signals into specific cellular responses within bacterial cells.
Automatically generated - may contain errors
Lab products found in correlation
C di amp, by Merck Group (4 mentions)
Cgamp, by Merck Group (2 mentions)
S epidermidis rp62a atcc 35984, by American Type Culture Collection (1 mentions)
Simplicity 185 system, by Merck Group (1 mentions)
Brain heart infusion, by BD (1 mentions)
Tryptic soy broth tsb, by BD (1 mentions)
High performance liquid chromatography (hplc), by Thermo Fisher Scientific (1 mentions)
2 3 cgamp, by Merck Group (1 mentions)
N methyl pyrrolidone (nmp), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
9 protocols using c di gmp
1
C-di-GMP Binding Assay Protocol
2
Fabrication and Characterization of SNAP-Loaded Thermoplastic Silicone Polyurethane Substrate
3
Murine Intranasal and Sublingual Immunization with H5N1 Virosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Groups of mice (three to five animals) were immunized either i.n. or s.l. on days 0 and 21 with PBS or isotonic saline (mock), or with H5N1 virosomes; the latter were administered alone or with different adjuvants—c-di-AMP, c-di-GMP, CTB (Sigma-Aldrich, Germany), last (or third) generation adjuvant (LGA) of immune stimulating complexes (Matrix M™)—made up to a maximal volume of 20 µl (i.n.) or 10 µl (s.l.) in PBS or isotonic saline solution.
4
Extraction and Quantification of c-di-GMP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extraction of c-di-GMP was performed as described previously [28 (link)], and quantification of extracted c-di-GMP was carried out by high performance liquid chromatography (Dionex). Commercially available c-di-GMP (Sigma-Aldrich) was used as a reference.
5
Synthesis and Purification of Labeled RNA Hairpins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All RNAs were synthesized by run-off in vitro transcription using T7 RNA polymerase as previously described (Stoldt et al. 1998 (link)). Unlabeled, 15N-adenine or 13C,15N-adenine labeled Vc2, Vc2 G20A, Gs1761, Gs1761WT as well as Cbe 1–2 were transcribed using SmaI linearized plasmid DNA or PCR generated double-stranded DNA fragments as templates and purified by denaturing PAGE as previously described (Duchardt-Ferner et al. 2010 (link)). 13C,15N- and 15N-labeled rNTPs were purchased commercially (Silantes).
RNAs were folded under conditions favoring monomeric hairpin structures in a low salt buffer (2.5 mM Bis-Tris, pH 6.5) by denaturing at 70°C for 5 min, rapid 10-fold dilution with the same ice-cold buffer and subsequent annealing on ice. They were exchanged into 25 mM Bis-Tris buffer, pH 6.5, 5 mM magnesium acetate for NMR spectroscopy using ultracentrifugation devices (VivaSpin 2, MWC 3 kDa) and multiple cycles of concentration and dilution with NMR buffer. The RNAs in the final NMR samples were monomeric and homogeneous as judged from a native gel (Supplemental Fig. S5 ).
Unlabeled c-di-GMP and c-GAMP were purchased commercially as sodium salts (Sigma Aldrich) and dissolved in NMR buffer.
RNAs were folded under conditions favoring monomeric hairpin structures in a low salt buffer (2.5 mM Bis-Tris, pH 6.5) by denaturing at 70°C for 5 min, rapid 10-fold dilution with the same ice-cold buffer and subsequent annealing on ice. They were exchanged into 25 mM Bis-Tris buffer, pH 6.5, 5 mM magnesium acetate for NMR spectroscopy using ultracentrifugation devices (VivaSpin 2, MWC 3 kDa) and multiple cycles of concentration and dilution with NMR buffer. The RNAs in the final NMR samples were monomeric and homogeneous as judged from a native gel (
Unlabeled c-di-GMP and c-GAMP were purchased commercially as sodium salts (Sigma Aldrich) and dissolved in NMR buffer.
6
HPLC for c-di-AMP Kinetics and Quantification
7
Quantification of Cyclic di-GMP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cyclic dimeric guanosine monophosphate was extracted as described previously (Roy et al., 2013 ) and quantified by high-performance liquid chromatography (Dionex, Sunnyvale, CA, United States). Commercially available c-di-GMP (Sigma-Aldrich) was used as a standard.
8
Dinucleotide Derivative Hydrolysis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dinucleotide derivatives, including c-di-AMP, c-di-GMP (Sigma, St Louis, MO, USA) pApA, and pGpG (Biolog, Hayward, CA, USA), were used as substrates in the hydrolysis reaction (20 μL) containing 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1.0 mM DTT, 1.0 mM MnCl2, and 100 µg/mL BSA. PyapApase (1.5 μM) was incubated with 0.075 mM dinucleotide derivatives at 70 °C for 10 min to verify its activity. After incubation, 1 μL of 500 mM EDTA was added to the reaction to inactivate PyapApase.
9
Crystallization of PlzA Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For crystallization trials, purified His-tagged PlzA (5 mg ml–1) with and without c-di-GMP (18 mM; Sigma, St. Louis, MO, USA) was incubated overnight at 4°C then centrifuged to remove any precipitate. Using a Gryphon liquid dispensing system (Art-Robbins, Sunnyvale,CA, USA ), proteins were screened against the sparse matrix crystallization screens Core I, II, III, IV, JCSG + (Qiagen, Hilden, Germany) in 96-well INTELLI-PLATE 96 (Art-Robbins) by the vapor diffusion method in sitting drop configuration. Two hundred nL of protein was mixed with 200 nL of well solution and plates were incubated at 18°C. PlzA coincubated with c-di-GMP formed plate-like crystals within a week in 0.1 M succinic acid (pH 7.0) and PEG 3350 (15%; w/v). Conditions were further optimized using 24-well Linbro plates (Jena Bioscience, Jena, Germany ) by adjusting the concentration of precipitant and incubation temperature. The best crystals based on appearance and diffraction properties appeared after 7 days of incubation. Crystals of PlzA derivatized with selenomethionine were obtained under the same conditions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!