Approximately twenty micrograms of tissue were cut into fragments and placed in 2.0 mL screw cap tube containing 10 mm glass beads (Biospec) in 500 μL of RIPA buffer (Sigma) supplemented with protease inhibitors (Roche, Burgess Hill, UK) were disrupted using an automated tissue homogenizer (Biddy Scientific) for two thirty second pulses. Supernatant was transferred to a clean tube and spun at 14 000 rpm in a refrigerated table top microcentrifuge. Twenty microliters of the supernatant was run on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (GE Healthcare) using a semi-dry transfer unit (Bio-Rad). The blot was blocked in 3% non-fat milk at ambient temperature for one hour on a rotating shaker before incubation with an antibody directed to the IBDV capsid protein VP2 (generated by the microbiological services of The Pirbright Institute) in a 1:500 dilution overnight at 5 °C with gentle agitation. The blot was rinsed and a secondary HRP conjugated anti-mouse antibody (Sigma-Aldrich) was used at 1:10 000 dilution for one hour at room temperature before rinsing and visualization using ECL solution (Millipore) and exposure to X-ray film (GE Healthcare).
1.0 mm glass beads
Manufactured by Biospec
Sourced in United States, Germany
1.0 mm glass beads are spherical glass particles with a consistent diameter of 1.0 millimeters. They are made of high-quality borosilicate glass material.
Automatically generated - may contain errors
Lab products found in correlation
2.0 ml screw cap tubes, by Sarstedt (2 mentions)
Mini beadbeater 24, by Biospec (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
X ray film, by GE Healthcare (1 mentions)
Semi dry transfer unit, by Bio-Rad (1 mentions)
Hrp conjugated anti mouse antibody, by Merck Group (1 mentions)
Ecl solution, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Gc 2010, by Shimadzu (1 mentions)
Bead beater, by Biospec (1 mentions)
Magmax total nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Magmax lysis binding solution, by Thermo Fisher Scientific (1 mentions)
0.1 mm glass beads, by Biospec (1 mentions)
7 protocols using 1.0 mm glass beads
1
VP2 Protein Immunoblotting Protocol
2
Cecum SCFA Analysis Protocol
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Cecum contents were diluted five times in ice-cold PBS. Then, 10 to 20 1.0 mm glass beads (BioSpec, United States) were added. Samples were homogenized on a vortex for 90 s. After centrifugation for 10 min (13.000 RPM) at 4°C, supernatants were collected and stored at −80°C until further analysis. SCFA levels were detected by gas chromatography (Shimadzu GC2010, Shimadzu Corporation, Japan).
3
Fecal DNA Extraction and PCR Detection
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Fecal samples were maintained frozen at −80 °C until processed. A 300-μg sample of feces was placed into 1 mL of PBS and mixed thoroughly by vortexing for ~3 min. Solids were removed by centrifugation for 30 s at 100× g. Avoiding solid material, 175 mL of the supernatant was transferred to a tube containing 232 µL of MagMax lysis/binding solution (Thermo Fisher) prepared in accordance with the manufacturer’s instructions. These tubes also contained ~125 µL of 1.0 mm glass beads (Biospec Products), and ~1258 µL of 0.1 mm glass beads (Biospec Products). Bacilli were disrupted by shaking in a bead beater (Biospec Products) for 5 min. The sample was clarified by centrifugation at 16,000× g for 3 min. Using 20 µL of the clarified preparation DNA was isolated using the MagMAX total nucleic acid isolation kit; (Thermo Fisher), as instructed by the manufacturer. The IS900 element was detected by PCR, which was performed as described above.
4
Yeast Cell Lysis and Protein Extraction
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Frozen AHA labeled cultures were thawed on ice and resuspended in 1.0 mL of 1× PBS before being transferred to a 2.0 mL screwcap microfuge tube (Fisher, Hampton, NH, USA). The cells were pelleted via brief centrifugation and then resuspended in 1× PBS containing 1× Halt Protease inhibitor cocktail (Pierce) followed by approximately 25% v/v 1.0 mm glass beads (Biospec, Bartlesville, OK, USA). The yeast cells were lysed with 4–5 cycles of 45 s full speed in a bead disruptor (Thermo Fastprep FP120 (Biospec Products, Bartlesville Oklahoma USA)) with at least 5 min of cooling on ice between cycles. Whole-cell lysates were obtained via centrifugation at 16,000 rpm at 4 °C for 10 min. The supernatant was removed, carefully so as to not disturb the pellet, and transferred to a clean microfuge tube, and protein concentrations were determined via BCA assays (Pierce) on replicate samples that were then equilibrated to the same protein concentrations with 1× PBS +protease inhibitor as the diluent.
5
Quantifying Intestinal Bacterial Colonization
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After the specified time points, fish were euthanized, the intestine of each fish was aseptically dissected, placed into homogenization tubes (2.0-mL screw-cap tubes; Sarstedt, Numbrecht, Germany) with 1.5 g of 1.0-mm glass beads (BioSpec Products, Inc., Bartlesville, OK) and 1 mL of 1X PBS, and held on ice. Homogenization tubes were loaded into a Mini-Beadbeater-24 (BioSpec Products, Inc.) and shaken at maximum speed for two 1-min cycles, with the samples being incubated for 1 min on ice after both the first and last cycles. Intestinal homogenates from each fish were diluted and plated for enumeration on LB agar plates with appropriate antibiotics and incubated overnight at 37° C. AIEC and EcN were each selected on appropriate antibiotic media. In each colonization assay, whole intestine from a single zebrafish was homogenized and the colonization data were calculated as cfu per zebrafish intestine. Any colonization above or equal to 102 cfu per intestine was taken as detectable colonization. Uninfected zebrafish gut homogenate (control fish) was also diluted and spread over all the antibiotic containing LB plates used in this work as a negative control. No colonies were observed after overnight incubation at 37° C
6
RNA Extraction from Retinal Tissue
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At 4 h postinfection, infected and uninfected mice were euthanized and the retinal tissue harvested. Retinal tissue was placed in a 1.5 ml screw-cap tube containing 500 μl Sigma Tri reagent and 500 μl 1.0 mm glass beads (Biospec Products, Bartlesville, OK). After homogenization at 5000 rpm for 60 s, the supernatant was transferred to a sterile, nuclease free tube containing 200 μl chloroform and then mixed, incubated on ice, and centrifuged. The supernatant was added to 500 μl isopropyl alcohol and placed at -80 °C for 2 h to precipitate RNA. After centrifugation at 14,000 rpm for 20 min at 4 °C, the supernatant was discarded, the pellet washed with 500 μl 75% ethanol, and vortexed to resuspend the pellet. Following centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant was discarded and the pellet dried and then resuspended in 25 μl nuclease-free water. The concentration and purity were checked on a Nanodrop spectrophotometer and if necessary DNA contamination removed using the TURBO DNA free kit per the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA).
7
Intestinal Microbiome Quantification
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At specified time points, fish were euthanized using tricaine as described above. Intestines were aseptically removed and placed in homogenization tubes (2.0 ml screw-cap tubes; Sarstedt, Nümbrecht, Germany) with 1.5 g of 1.0 mm glass beads (BioSpec Products, Inc., Bartlesville, OK) and 1 ml of 1x PBS, and held on ice. Homogenization tubes were loaded into a Mini-Beadbeater-24 (BioSpec Products, Inc.). Serial dilutions of homogenized tissue were performed using 1X PBS and the dilutions were plated onto LB agar plates with appropriate antibiotics.
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