For protein extractions, femurs and tibias were isolated and cleaned of excess tissue, followed by surgical removal of the epiphyses, and removal of the marrow by centrifugation. The bones were then washed with cold PBS three times before being snap-frozen in liquid nitrogen, and subsequent pulverization in a Mikro Dismembrator U (Sartorius, Gottingen, Germany). Protein was extracted with 150 µL radioimmunoprecipitation assay (RIPA) buffer (cat# R0278; Sigma-Aldrich) containing phosphatase inhibitors (cat# 04906845001; Roche, Nutley, NJ, USA) and proteinase inhibitor (cat# 11836170001; Roche) on ice for 30 min. Protein concentration was measured with BCA assay (Thermo Scientific, Waltham, MA, USA), and Western blotting was performed according to standard protocols.
Mikro dismembrator u
Manufactured by Sartorius
Sourced in Germany
The Mikro-Dismembrator U is a laboratory equipment designed for the efficient disruption and homogenization of various sample materials. It utilizes a grinding ball to pulverize and homogenize the samples, preparing them for further analysis or processing.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Rneasy minelute spin column, by Qiagen (2 mentions)
Superscript 4 vilo master mix, by Thermo Fisher Scientific (2 mentions)
Quantstudio 6 flex system, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Rneasy mini spin column, by Qiagen (1 mentions)
Deoxyribonuclease 1 treatment, by Qiagen (1 mentions)
Phase lock gel, by Quantabio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Quant it dsdna broad range assay kit, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Trizma base, by Merck Group (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Chloroform, by Honeywell (1 mentions)
12 protocols using mikro dismembrator u
1
Bone Protein Extraction and Analysis
2
Quantitative Analysis of Tendon Gene Expression
3
Murine Cartilage Gene Expression Analysis
4
Quantifying Cilium and Hh Signaling Genes
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The supraspinatus tendon and enthesis were harvested, frozen in liquid nitrogen, and pulverized in a ball mill homogenizer (Mikro-Dismembrator U, Sartorius). Total RNA was extracted by TRIzol reagent and Invitrogen PureLink RNA Mini kit, as described in our previous study (2 (link)). The High-Capacity cDNA Reverse Transcription Kit (Invitrogen) was used to reverse transcribe RNA into cDNA. The relative abundance of cilium- and Hh signaling–related genes was evaluated by SYBR Green-based quantitative reverse transcription polymerase chain reaction (RT-PCR) on an Applied Biosystems QuantStudio 6 flex system. The sequences of RT-PCR primers used here are listed in table S1. Glyceraldehyde 3-phosphate dehydrogenase serves as housekeeping gene, and the relative mRNA expression level for each target gene was determined as 2−ΔΔCt.
5
Genomic DNA Extraction and Fragmentation
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Genomic DNA was isolated from a 5-day-old culture of alphaΔssgB7 (link) using phenol:chloroform extraction10 . Briefly, the cell pellet was resuspended in 10.3% (w/v) sucrose containing 0.01 M ethylenediamine tetraacetic acid (EDTA, 20296.291, VWR Chemicals BDH) pH = 8, following lysis with 10% (w/v) sodium dodecyl sulfate (SDS, 20765.02, Serva). Extraction with phenol:chloroform (1:1 mix of phenol, 10001173, Fisher BioReagentstm and chloroform, 32211, Honeywell) was performed and the nucleic acids were precipitated using isopropanol (33539, Honeywell). The pellet was dissolved in Tris-EDTA buffer (Trizma® base, RDD008, Sigma-Aldrich), followed by RNase A (EN0531, Thermo Fisher) and Proteinase K treatment (19131, Qiagen). The gDNA was isolated using phenol:chloroform extraction and precipitated using absolute ethanol (5250501, Biosolve) before resuspension in nuclease-free water. Fragmented gDNA was obtained by beat-beating the intact gDNA for 12 min using 2 mm diameter glass beads in a Mikro-Dismembrator U (Sartorius) at 2000 rpm. Chromosomal DNA concentrations were verified using the Quant-IT™ Broad-Range dsDNA Assay Kit (Q33130, Invitrogen).
6
Mouse Muscle Nuclear Extraction
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For mouse muscle nuclear extracts, frozen tissues were disrupted using a Mikro-Dismembrator U (Sartorius, Göttingen, Germany) followed by a Dounce homogenizer in Buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl) with freshly added 1 mM DTT and protease inhibitor cocktail (Complete, Roche, Basel, Switzerland). Myoblasts were pelleted at 300xg for 10 min at 4°C and washed with cold PBS. Cells were resuspended in Buffer A and incubated 15 min on ice. Cell samples were vortexed in pulsed for 10 min and tissue samples were vortexed for 10 seconds in 0.5% NP-40. Nuclei were pelleted at 4500xg for 20 seconds, the cytoplasmic fraction was removed, and nuclei were washed with Buffer C (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA and 10% glycerol) including protease inhibitors (Complete, Roche) and phosphatase inhibitors (10 mM NaF, 10 mM β-glycerophosphate, 1 mM sodium vanadate). Pellets were resuspended in Buffer C and vigorously shaken on ice for 30 min followed by centrifugation at 14 000xg for 10 min at 4°C. The supernatant, which is the nuclear extract fraction, was collected. Protein concentrations were determined by standard BCA assays (Pierce, Waltham, MA, USA). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
7
Protein Expression Profiling using RPPM
8
Rapid Brain Extraction and Homogenization
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Immediately after the last behavioral test, the animals were placed individually for 10 min in a glass dish containing an ice water immersion (2–4 °C), then rapidly euthanized, as previously reported by Brinza et al. [34 (link)]. Whole brains were carefully excised, weighed, and placed in 0.5 mL tubes, and stored at −20 °C until further use.
The next day, the brains of fish from the same batch of animals were weighed individually (~3–6 mg) and homogenized in extraction phosphate buffer solution (0.1 M potassium phosphate buffer solution, pH 7.4 with KCl 1.15%) at a ratio of 1 to 10 using a ball mill (Mikro-Dismembrator U; Sartorius, NY, USA). The homogenate was centrifuged (15 min at 14,000 rpm) and the supernatant was later used for the determination of biochemical parameters.
The next day, the brains of fish from the same batch of animals were weighed individually (~3–6 mg) and homogenized in extraction phosphate buffer solution (0.1 M potassium phosphate buffer solution, pH 7.4 with KCl 1.15%) at a ratio of 1 to 10 using a ball mill (Mikro-Dismembrator U; Sartorius, NY, USA). The homogenate was centrifuged (15 min at 14,000 rpm) and the supernatant was later used for the determination of biochemical parameters.
9
Intestinal Gene Expression Analysis
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The intestinal fragments that were freshly collected and immediately frozen in liquid nitrogen were stored at -170°C until processing to study the expression of the c-fos and c-jun genes in association with cell proliferation and apoptosis. First, total RNA was extracted from intestinal samples using TRIZOL™ reagent (Invitrogen, Carlsbad, CA, USA). Approximately 100 mg of intestine was fragmented in a tissue mill (Mikro Dismembrator U, Sartorius AG, Goettingen, Germany) after the addition of liquid nitrogen and was then homogenized in 700 μl of TRIZOL solution according to the manufacturer’s protocol.
The total RNA concentration was measured using a spectrophotometer (BioPhotometer, Eppendorf AG, Germany) at 260 nm, and RNA quality was assessed by calculating the 260 nm/280 nm absorbance ratio. One microgram of RNA was subjected to agarose gel electrophoresis to assess its integrity by visualizing the fragments corresponding to 18S and 28S ribosomal RNAs.
Complementary DNA (cDNA) was prepared from 2 μg of total RNA via reverse transcription using 200 U of SuperScript III RNase H-RT (Invitrogen) and oligo(dT)s as primers. The resulting cDNA solution was stored at -20°C.
Gene expression was evaluated by two methods, conventional semiquantitative RT-polymerase chain reaction (PCR) and real-time RT-PCR analyses, and the results of these two methods were compared.
The total RNA concentration was measured using a spectrophotometer (BioPhotometer, Eppendorf AG, Germany) at 260 nm, and RNA quality was assessed by calculating the 260 nm/280 nm absorbance ratio. One microgram of RNA was subjected to agarose gel electrophoresis to assess its integrity by visualizing the fragments corresponding to 18S and 28S ribosomal RNAs.
Complementary DNA (cDNA) was prepared from 2 μg of total RNA via reverse transcription using 200 U of SuperScript III RNase H-RT (Invitrogen) and oligo(dT)s as primers. The resulting cDNA solution was stored at -20°C.
Gene expression was evaluated by two methods, conventional semiquantitative RT-polymerase chain reaction (PCR) and real-time RT-PCR analyses, and the results of these two methods were compared.
10
Strep-Tactin Affinity Purification of GST-Tagged Proteins
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Strep-Tactin®-coated beads (MagStrep type3 XT, IBA) were incubated with equal molar amounts of the GST-Twin-Strep-tagged proteins (0.2 nmol) for 1 h at 4°C in a final volume of 250 μl of CLB. Then the beads were washed with chilled CLB. Mouse hearts were powdered using a Mikro-Dismembrator U (Sartorious). Proteins were extracted with CLB supplemented with 1× protease inhibitor cocktail and 1 mM EDTA by vortexing on ice. Insoluble components were removed by centrifugation at 16,000 g for 15 min at 4°C. The lysates were incubated for 3 h at 4°C in a rotating mixer with the GST-Twin-Strep-tagged proteins previously bound to the Strep-Tactin®-coated beads. Following incubation, samples were washed using CLB, eluted with 2× Laemmli buffer and boiled for 5 min at 95°C or incubated at RT for 45 min and then resolved by SDS-PAGE and analyzed by western blot or mass spectrometry.
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