We prepared a simple agar-based medium (one g agar in 100 ml distilled water) containing five mg ml−1Micrococcus lysodeikticus (lyophilized Micrococcus lysodeikticus, ATCC No. 4698, Sigma-Aldrich; note that Micrococcus lysodeikticus and Micrococcus luteus are considered to be the same group of bacteria, e.g. Litman, 1968 (link)) cell wall residues homogeneously distributed, and 0.6 mg ml−1 ampicillin sodium. This strain was used because of its commercial availability. The basic principle of this method is that samples containing lysozyme will produce transparent clearing zones in the opaque layer of cell wall containing agar, and the area of clearing zones is proportional to the lysozyme concentration of the sample. We poured five ml of this medium in each petri dish and applied two μl from each sample on the surface of the cell wall agar, in duplicates from each spider. Duplicates were placed in the same petri dish in the case of each sample, and in one petri dish 10 samples (i.e. five duplicates) were applied. Petri dishes were then incubated at 30 °C for 24 h. Following the incubation, all petri dishes were photographed, and the areas of clearing zones were measured using ImageJ (see below).
Micrococcus lysodeikticus
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
Micrococcus lysodeikticus is a Gram-positive bacterium commonly used as a substrate for assaying lysozyme activity. It is a lyophilized powder that can be reconstituted in water or buffer solutions.
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Lab products found in correlation
Hen egg white lysozyme, by Merck Group (7 mentions)
Imark microplate absorbance reader, by Bio-Rad (3 mentions)
Lysozyme, by Merck Group (2 mentions)
Glycine, by Merck Group (2 mentions)
Mueller hinton agar plate, by bioMérieux (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Azobisisobutyronitrile, by Fujifilm (1 mentions)
Lysozyme from chicken egg white, by Merck Group (1 mentions)
2 dodecylthiocarbonothioylthio 2 methylpropionic acid, by Merck Group (1 mentions)
Thioflavin t tht, by Merck Group (1 mentions)
3h adomet, by Cytiva (1 mentions)
Model 550 microplate reader, by Bio-Rad (1 mentions)
N phenylthiourea ptu, by Merck Group (1 mentions)
Uv 1600pc, by Shimadzu (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Elisa plate reader, by Bio-Rad (1 mentions)
Rnase a, by Merck Group (1 mentions)
Nitrocellulose filter, by Merck Group (1 mentions)
Cystine hydrochloride, by Merck Group (1 mentions)
90 protocols using micrococcus lysodeikticus
1
Lysozyme Activity Quantification Assay
2
Tear Lysozyme Concentration Measurement
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The tears were collected by gently applying a 5-mm-diameter filter paper disc in the inferior conjunctival cul-desac of both eyes for 1 min, with the eyes closed. The samples were stored at -20°C until they were processed. To determine tear lysozyme concentration, we performed the Micrococcus lysodeikticus (ATCC 4698, 770; Sigma-Aldrich, St. Louis, MO) agar diffusion assay in Mueller Hinton agar plates (Bio Merieux, Marcy l'Etoile, France). Each disc was placed on the plate containing the Micrococcus lysodeikticus (2 × 106 CFU/mL) suspension gel, and the inhibition halo was measured after 24 h. To calculate the lysozyme concentration, a standard curve was obtained using identical discs dampened with 10000, 1000, 100, and 10 mg/mL of lysozyme (ATCC 4698, L6876; Sigma-Aldrich) diluted in phosphate-buffered saline (Invitrogen Corp., Carlsbad, CA). Values £1000 mg/mL were considered abnormal (22 22 González-García MJ, González-Sáiz A, de la Fuente B, Morilla-Grasa A, Mayo-Iscar A, San-José J, et al. Exposure to a controlled adverse environment impairs the ocular surface of subjects with minimally symptomatic dry eye. Invest Ophthalmol Vis Sci. 2007;48(9):4026-32. ) .
3
Quantifying Nasal Lysozyme Levels
4
Proteolytic and Lytic Activities of VgrG2b
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Generic protease activity of 10 μg recombinant VgrG2bC-ter, its inactive mutant and a proteinase K control was assessed using 1% proteinaceous substrates bovine serum albumin and gelatin in 2% bacteriological agar plates. After incubation at 37°C for 24 h, plates were stained with amido black solution (Vermelho et al., 1996 (link)) to visualize zones of proteolysis. Lysozyme activity of recombinant proteins utilized the lyophilised substrate Micrococcus lysodeikticus (Sigma Aldrich) as previously documented (Lossi et al., 2011 (link)). The polymyxin B-mediated permeabilisation of the bacterial outer membrane to permit access of exogenous proteins to the periplasmic space was performed as described elsewhere (Brooks et al., 2013 (link)), except E. coli was employed. E. coli DH5α was resuspended in turbidometry buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl) at OD600 0.2 and incubated with 4 μg/ml polymyxin B. After addition of 10 μg purified protein, the turbidity at OD600 was monitored at five-minute intervals for 1 h.
5
Turbidometric Serum Lysozyme Assay
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Serum lysozyme (Lyso) was assessed using the turbidometric assay (Ellis 1990 ). A suspension of 875 μL of Micrococcus lysodeikticus (Sigma, ATCC 4698) at a concentration of 0.2 mg mL−1 (in PBS) and was added to 25 μL of serum samples were measured spectrophotometrically at 530 nm after 0.5 and 4.5 min at 25 °C, using a spectrophotometer. A unit of lysozyme activity was defined as the amount of serum causing a reduction in absorbance of 0.001 min−1.
6
Synthesis and Characterization of Sulfobetaine-Based Polymers
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Sulfobetaine (SPB) monomer was donated by Osaka Organic Chemical (Osaka, Japan) and used without further purification. 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid, Thioflavin T (ThT), Micrococcus lysodeikticus, and lysozyme from chicken egg white were purchased from Sigma-Aldrich. Azobisisobutyronitrile (AIBN) was purchased from Wako Pure Chemical Industries (Osaka, Japan) and was recrystallized from methanol before use. Ethylene glycol dimethacrylate, BuMA and 4,4′-azobis-(4-cyanovaleric acid) (V-501, initiator) were purchased from TCI (Tokyo, Japan). Prior to its use, the inhibitor from BuMA was removed by passing through an inhibitor removal prepacked column (Sigma-Aldrich).
7
Purification and Activity of DNA Methyltransferases
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For overexpression and purification of the putative DNA-specific methyltransferases, the open reading frames (ORFs) were amplified from chromosomal DNA by PCR using primers for the directed in-frame cloning with the N-terminal His-tag into vectors of the pBAD/His A, B, C series (Invitrogen). Correct insertions were verified by sequencing. For overexpression, recombinant cells of the methyltransferase-defective E. coli strain TOP10 were cultured at 30°C in LB medium. The expression of the recombinant protein was induced at OD500 = 1.0 by addition of arabinose (0.002% final concentration). The proteins were extracted from E. coli by sonication, and the fusion proteins were purified on a Ni-NTA matrix (ProBondTM Resin, novex, life technologies) after elution with an imidazole gradient. The resulting protein fractions were evaluated using SDS gels containing 12% acrylamide. The recombinant proteins were detected by immuno-blotting with an antibody specific for the N-terminal His-tag (Invitrogen).
The DNA-specific methyltransferase activity was assayed by incubation of non-methylated chromosomal DNA of Micrococcus lysodeikticus (Sigma) with [3H]-AdoMet (Amersham), as described.20 (link) The protein content was estimated according to reference.31 (link)
The DNA-specific methyltransferase activity was assayed by incubation of non-methylated chromosomal DNA of Micrococcus lysodeikticus (Sigma) with [3H]-AdoMet (Amersham), as described.20 (link) The protein content was estimated according to reference.31 (link)
8
Quantifying Lysozyme Activity in Sea Urchin Fluids
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lysozyme activity was quantified in both CFC and CL, according to Santarém et al. (1994 ) and Fernández-Boo et al. (2018 (link)). Coelomic fluid from each sea urchin was centrifuged at 780g for 10 min. The supernatant, corresponding to CFC, was collected, whereas the coelomocytes were resuspended in distilled water and sonicated at 4 °C for 1 min to obtain CL. CL and CFC were frozen and stored at − 80 °C before analyses. Fifty microlitres of CL and CFC were added to 950 μl of a 0.15% suspension of Micrococcus lysodeikticus (Sigma) in 66 mM phosphate buffer, pH 6.2; and the decrease in absorbance (ΔA min−1) was continuously recorded at 450 nm for 5 min at room temperature. Standard solutions containing 1, 2.5, 5 and 10 μg lysozyme per ml of 66 mM phosphate buffer, pH 6.2, were prepared from crystalline hen egg-white lysozyme (Sigma). The average decrease in absorbance per minute was determined for each enzyme solution, and a standard curve of enzyme concentration versus ΔA min−1 was drawn. Results were expressed as μg lysozyme mg protein−1. CL and CFC protein concentrations were also quantified according to Bradford (1976 (link)).
9
Plasma Lysozyme Activity Quantification
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The lysozyme activity was measured in plasma samples with a standard turbidity test described by Keller et al. [52 (link)], modified. Briefly, 1 mg/mL stock solution of hen egg white lysozyme (HEL, Sigma, St. Louis, MO, USA) was prepared in 0.1M phosphate buffer (pH 5.9) and serially diluted in phosphate buffer to produce the standard curve of 0, 0.3, 0.6, 1.25, 2.5, 5, 10, 20, and 25 μg/mL. To obtain a fresh solution of Micrococcus lysodeikticus (Sigma, St. Louis, MO, USA), 50 mg of the lyophilized cells were dissolved in 0.1 M phosphate buffer. Each concentration of the standard curve (25 μL/well) was added to a 96-well plate in triplicate, and 25 μL of each sample was added in quadruplicate to the same plate. The M. lysodeikticus solution (175 μL/well) was quickly added to three sample wells and to each of the standard wells. The blank was the fourth well containing plasma with 175 μL of phosphate buffer without M. lysodeikticus. Absorbance at 450 nm was measured using a microplate ELISA reader (Microplate Reader Model 550, Bio-Rad). The optical density (O.D.) was measured immediately (T0) and after 5 min (T5) and the activity expressed as HEL concentration (μg/mL) by linear regression of the standard curve.
10
Quantifying Lysozyme Activity in Hemolymph
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The lysozyme (LYS) activity was quantified following the procedures described by Chiu et al. (2007) (link). Briefly, 500 μl of diluted hemolymph were centrifuged, and the precipitate was mixed with 1 ml (0.02%) of Micrococcus lysodeikticus (Sigma, St. Louis, MO, United States). The reaction was carried out at room temperature, and the absorbance at 530 nm was measured after 0.5 and 4.5 min. A unit of LYS activity was defined as the amount of enzyme producing a decrease in absorbance of 0.01 min–1, and the specific activity was expressed as U (g protein–1).
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