The elastolytic activity of P. aeruginosa was determined as previously described15 (link) using Elastin Congo Red (ECR, Sigma) as the substrate.
Elastin congo red ecr
Manufactured by Merck Group
Sourced in United States
Elastin-Congo red (ECR) is a lab equipment product that is used to detect and quantify the presence of elastin in biological samples. It functions by binding to elastin, a key structural protein found in connective tissues, and producing a color change that can be measured using spectrophotometric techniques.
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Lab products found in correlation
8 protocols using elastin congo red ecr
1
Elastolytic Activity of P. aeruginosa
2
Elastolytic Activity of P. aeruginosa
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The elastolytic activity of the cell-free culture supernatant of P. aeruginosa was determined by following the method of Ohman et al.16 (link) by using Elastin-Congo red (ECR; Sigma, St. Louis, USA) as the substrate. One hundred (100) μl of (only vitexin as well as in combination) treated and untreated P. aeruginosa culture supernatant was mixed with 900 μl of ECR buffer (100 mM Tris, 1 mM CaCl2, pH 7.5) containing 20 mg of ECR and incubated at 37 °C for 3 h. The reaction was terminated by adding 1 ml of 0.7 M sodium phosphate buffer (pH 6.0) and the tubes were placed in cold water bath. The insoluble ECR was removed by centrifugation at 10,000 rpm for 10 min and then the absorbance was measured at 495 nm.
3
Evaluating Pomegranate Elastase Inhibition
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The estimation of the effects of P. granatum extracts on the elastolytic activity of P. aeruginosa PAO1 was established based on protocols published by Vasavi et al. (24 (link)). Elastin Congo red (ECR; Sigma, St. Louis, USA) assay was used to measure elastase activity. PAO1 was grown in Luria-Bertani broth (LB; Sigma Aldrich, USA) at 37°C and the different concentrations of methanolic extracts (0.5; 1; 2.5; 5 and10 mg/ml). PAO1 culture was centrifuged and 100 μl supernatant was added to 900 μl of Elastin Congo Red buffer (100 mM Tris, 1 mM CaCl2, pH 7.5) containing 20 mg of elastin Congo red and incubated with shaking at 37°C for 3 h. After centrifugation for 15 min at 13,000 rpm, 200 μl of the supernatant were recovered in 96-well flat-bottom ELISA plates. The optical density was measured at 495nm using a specific ELISA reader (France).
4
Elastase Activity Assay in Biofilms
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Elastase activity was measured in cell-free supernatants from SM23 Pseudomonas cells, treated or not with the compound (concentration range from 0.780 to 3.125 μM), during biofilm formation. After 6, 12, and 24 h of culture, the elastase activity was measured as described by (Ohman et al., 1980 (link)) using Elastin-Congo Red (ECR) (Sigma, St. Louis, USA) as a substrate. Briefly, 100 μl of untreated or SM23-treated supernatants were mixed with 900 μl of ECR buffer (100 mM Tris, 1 mM CaCl2, pH 7.5) containing 20 mg of ECR and then incubated for 3 h at 37°C. The reaction was terminated by adding 1 ml of 0.7 M sodium phosphate buffer (pH 6.0) and the tubes were placed in cold water bath. The insoluble ECR was removed by centrifugation at 10,000 rpm for 10 min and then the absorbance was measured at 495 nm by a SunRise Microplate Reader. The elastase activity was expressed as the optical density (OD495) mean ± SEM.
5
Elastase Inhibitor Activity Assay
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Synthetic elastase inhibitor was supplied by the pharmaceutical laboratory of Sun-Yat-sen University according to the procedure as described.13 (link) The ability of elastase inhibitor to inhibit the proteolytic activity of elastase was tested using Elastin-Congo red (ECR; Sigma-Aldrich, St. Louis, MO, USA) as the substrate. 100 µl elastase inhibitor was added to the 900 µl mixture of 2 µg/ml exoproteins and ECR buffer (20 mg ECR, 100mM Tris-HCL, pH 7.5), then incubated for 18 hours at 37°C. The liquid supernatant was collected to measure the absorbance value at 495 nm after centrifugation at 16 000 g for 15 minutes. The elastase activity was expressed as the ratio of the OD495 nm and OD600 absorbance value.
6
Elastolytic Activity of P. aeruginosa Culture Supernatant
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The elastolytic activity of the cell-free culture supernatant of P. aeruginosa was determined using Elastin-Congo red (ECR; Sigma) as the substrate [26 (link)]. 100 μL supernatants of P. aeruginosa incubated in different concentrations of glucose (0, 50, 100, 150 and 200 mg/mL) for 17 h were added to 900 μL of ECR buffer (100 mM Tris, 1 mM CaCl2, pH 7.5) containing 10 mg of ECR and incubated at 37 °C for 3 h. The reaction was terminated by adding 1 mL of 0.7 M sodium phosphate buffer (pH 6.0) and the tubes were placed in a cold water-bath. The insoluble ECR was removed by centrifugation at 10,000 rpm for 10 min, and the absorbance was measured at OD495.
7
Quantifying Pseudomonas Elastase Activity
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The elastase activity of all P. aeruginosa strains was determined using Elastin-Congo red (ECR; Sigma-Aldrich). Briefly, for each sample, 100 μL concentrated filter-sterilized bacterial exoprotein was added to 900 μL of ECR buffer (100 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 20 mg ECR) and incubated for 18 h at 37°C with agitation at 200 rpm. Then insoluble ECR was removed by centrifugation at 16,000 g for 15 min at room temperature, and the absorbance was measured at 495 nm. Fresh LB medium was used as the blank control, and LB with ECR as the negative control. Secreted elastase activity was expressed as the ratio of the OD495 and OD600 absorbance value.
8
Bacterial Elastase Activity Quantification
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The elastase activity was determined using Elastin–Congo red (ECR; Sigma) as the substrate. Briefly, 10 mg of ECR was introduced to 100 μL of the bacterial culture supernatant and 900 μL of ECR buffer (100 mM Tris, 1 mM CaCl2; pH 7.5), followed by incubation at 37°C for 18 h. Elastase, produced by bacteria, can break down elastin. After the reaction was completed, 1 mL of 0.7 M sodium phosphate buffer (pH 6.0) was added and the reaction mixture was placed in the cold water bath to stop the reaction. The undecomposed elastin was separated through centrifugation, and the supernatant absorbance value was recorded at 498nm. The experiment was repeated thrice, and the average value was calculated.
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