Cell pellets were homogenized on a FastPrep-24 instrument using the lysis matrix B (MP Biomedicals, Solon, OH) for 4 repeated 40-s cycles at 6.5 m/s in 200 μl of the buffer containing 2% sodium dodecyl sulfate and 50 mM triethylammonium bicarbonate (TEAB). Samples were centrifuged at 6,200g for 20 min. The supernatants were transferred to clean tubes. The lysis tubes were washed with 110 μl of the lysis buffer and centrifuged again. The supernatants were combined with the corresponding lysates from the previous step. Protein concentration in the combined lysates was determined using Pierce BCA Protein Assay Kit (Thermo Scientific, Ref. 23225) and the Benchmark Plus microplate reader (BIO-RAD) with bovine serum albumin solutions as standards.
Lysis matrix b
Manufactured by MP Biomedicals
Lysis matrix B is a specialized laboratory product designed for cell lysis. Its core function is to facilitate the efficient disruption of cell membranes, enabling the release of cellular contents for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Benchmark plus microplate reader, by Bio-Rad (1 mentions)
Fastprep, by MP Biomedicals (1 mentions)
Fastprep 24 apparatus, by MP Biomedicals (1 mentions)
Sw41ti rotor, by Beckman Coulter (1 mentions)
Tissuelyser bead beater, by Qiagen (1 mentions)
Dry bath system, by Starlab (1 mentions)
Magna pure compact machine, by Roche (1 mentions)
Bacterial lysis buffer, by Roche (1 mentions)
Magna pure compact na isolation kit 1, by Roche (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Mouse anti his, by Thermo Fisher Scientific (1 mentions)
Precellys evolution homogenizer, by Bertin Technologies (1 mentions)
4 protocols using lysis matrix b
1
Protein Extraction from Cell Pellets
2
Ribosome Profiling of S. aureus
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S. aureus USA300 and NE1546 strains were cultivated in 20 mL BHI medium at 37°C until OD600 nm 2.0. Cells were resuspended in 500 µL lysis buffer (20 mM Tris pH 8, 100 mM NH4Cl, 50 mM MgCl2, 0.4% Triton X-100, 0.1% nonidet P-40), transferred into a tube with lysis matrix B (MP Biomedicals), and lyzed by bead beating in a FastPrep-24 apparatus (MP Biomedicals) at 6 m/sec for 40 sec. Cell debris was removed by centrifugation (16,000g for 10 min at 4°C) and the supernatant was recovered. Equal OD260 nm units of each sample were layered on top of 5%–50% sucrose gradient prepared in buffer G (20 mM Tris pH 8, 100 mM NH4Cl, 15 mM MgCl2) and separated by ultracentrifugation in a Beckman SW-41Ti rotor at 39,000 rpm for 2 h 46 min at 4°C. Finally, samples were fractionated on a piston gradient fractionator (Biocomp), and the OD260 nm was recorded to generate sedimentation profiles.
3
Efficient DNA Extraction from Bone Powder
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DNA was extracted from the bone powder received from UoS using a bead-beating approach. Initially, 700 μl bacterial lysis buffer (Roche UK- Cat. No. 4659180001) was added to the powder and vortexed before being transferred to a bead beating tube (Lysis Matrix B, 2ml tube (116911050, MP Biomedicals). The sample was bead-beaten for 2 minutes at full speed on a Tissue Lyser bead beater (Qiagen- Cat. No. 69980) and then pulse centrifuged (Eppendorf UK centrifuge model 5424). An aliquot (420 μl) was then transferred to a fresh Eppendorf tube and 20 μl of proteinase K (>600mAu/ml) (Qiagen, Cat. No. 19133) was added. The sample was then incubated at 65°C on a dry bath system (StarLab UK) for 10 minutes. Finally, DNA was purified on the MagNA Pure Compact machine (Roche UK) using the Roche MagnaPure Compact DNA_bacteria_V3_2 protocol (MagNA pure compact NA isolation kit I, Roche UK- product 03730964001).
4
Stability of Recombinant Proteins at Varying Temperatures
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Aliquots of lyophilized sp79 and sp82 biomass were stored in sealed moisture-free chambers away from light for one year at −80 °C, −20 °C, 4 °C, 25 °C, 37 °C and 42 °C. Samples were brought to 1 mg/mL in SDS sample buffer, boiled for 5 min at 100 °C, and stored in −80 °C until analysis by SDS-PAGE followed by western blot with rabbit anti-Myc antibody (Rockland) at 1:3000 and donkey anti-rabbit (Invitrogen) at 1:10,000. ImageJ software was used to estimate band intensity relative to a sample taken to represent time zero (from fresh harvest). For sp648, biomass samples were processed and stored under identical conditions as above for 10 months. To harvest the total soluble protein, measured aliquots were resuspended in PBS with Halt Protease Inhibitor and lysed on a Precellys Evolution Homogenizer (Bertin Instruments) using Lysis Matrix B (MP Bio) beads, followed by centrifugation to collect the supernatant. Automated immunoblotting was done via the JessTM Simple Western nano-immunoassay system (ProteinSimple, San Jose, CA, USA), following the manufacturer’s protocol, using mouse anti-His (ThermoFisher Scientific) followed by goat anti-mouse HRP conjugate (ProteinSimple) and rabbit-NANP (Alpha Diagnostic) followed by goat anti-rabbit HRP conjugate (ProteinSimple).
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