For METTL20 target screening, HEK293T cells were cultured for at least six cell doublings in DMEM supplemented with 10% of dialysed FCS containing either a light (12C6) or stable heavy (13C6) isotope in lysine after processing with the SILAC labelling kit (Pierce, cat. # 89983). The mitochondrial fractions of each cell type were purified as described previously28 (link). Briefly, cell pellets were resuspended in SEM buffer (10 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES]-KOH pH 7.5, 0.2 M mannitol, 50 mM sucrose, 10 mM KCl, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride), and homogenised by means of the Dounce tissue grinder (2 ml, Pestle A, Fisher Scientific, USA). After that, the supernatants were centrifuged at 700× g for 10 min to separate nuclear pellets from the supernatants containing cytosolic materials and mitochondria. The supernatants were again centrifuged at 700× g to remove residual pellets, and then at 7,000× g for 10 min to separate the mitochondrial pellet from the cytosol-enriched supernatant. The mitochondrial pellets were next washed twice with SEM buffer, and the resulting mitochondrial fractions were stored at −80 °C.
Dounce tissue grinder
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dounce tissue grinder is a laboratory instrument used for the mechanical disruption and homogenization of soft tissue samples. It consists of a glass vessel with a tight-fitting glass pestle, allowing for the gentle and controlled breakdown of tissue samples in a variety of buffer solutions.
Automatically generated - may contain errors
Lab products found in correlation
Rnase t1, by Thermo Fisher Scientific (2 mentions)
Qubit assay, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rnase h, by Thermo Fisher Scientific (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Surepage bis tris gel, by GenScript (2 mentions)
Protease inhibitor cocktail, by Mei5 Biotechnology (2 mentions)
Chemiscope 6300, by Clinx (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
6 protocols using dounce tissue grinder
1
METTL20 Target Screening in HEK293T Cells
2
Preparation of Membrane Vesicles from HEK293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For preparation of HMVs, HEK293 cells expressing GFP-CAAX were trypsinized using 0.05% trypsin-EDTA (Gibco) and harvested by centrifugation at 300 × g for 5 min. The cells were resuspended in 1 mL of a solution of sucrose (20%) and Tris·HCl (30 mM, pH 8.0) and homogenized using 1 mL of Dounce tissue grinder (Fisher Scientific). Cells were spun at 4 °C for 5 min at 7,500 × g, and the supernatant was transferred to a fresh microcentrifuge tube to isolate the membranes from the cytoplasmic fraction and cell debris. This supernatant was then spun at 4 °C for 30 min at 40,000 × g. The membrane fraction was obtained as a pellet following the high-speed centrifugation, and the supernatant was discarded. The membrane vesicles were resuspended in 500 μL of buffer composed of Tris·HCl (30 mM, pH 8.0), sucrose (20%), and EDTA (0.1 mM). For BMVs, cells were grown and collected as described above. Frozen cells were processed and membrane vesicles were prepared as outlined, using previously reported protocols (23 (link)).
3
Tissue Extraction and Nucleic Acid Purification
4
Tissue Extraction and Nucleic Acid Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue derived from the ILPFC of retention control (RC) or extinction (EXT) trained mice was homogenized by Dounce tissue grinder in 500 μl cold 1X PBS (Gibco). 400 μl of homogenate was used for DNA extraction, and 100 μl was used for RNA extraction. DNA extraction was carried out using DNeasy Blood & Tissue Kit (Qiagen) with RNAse A (5 prime), RNAse H and RNAse T1 treatment (Invitrogen), and RNA was extracted using Trizol reagent (Invitrogen). Both extraction protocols were conducted according to the manufacturer’s instructions. The concentration of DNA and RNA was measured by Qubit assay (Invitrogen).
5
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell samples were washed with PBS 3 times and collected; brain tissues were ground 20–30 times using Dounce Tissue Grinders (Thermo Fisher, K8853000002). Then the samples were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 0.1% SDS, 0.5% DOC, and 1% Triton X-100) with Protease inhibitor cocktail (Mei5bio, MF182-plus-10), and sonicated 6 times. The protein in the samples was separated by SDS-PAGE electrophoresis using SurePAGE™ Bis-Tris Gels (GenScript, M00652), and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% skim milk at room temperature for 1 h and incubated with the corresponding primary antibodies in 3% BSA at 4 °C overnight. The next day, the membranes were washed three times with 1× PBS and incubated with HRP-conjugated secondary antibodies in 5% skim milk at room temperature for 1 h. The signals were developed with ECL solution (Millipore, WBKLS0500) after washing three times in 1 X PBS. The images were acquired digitally using Clinx ChemiScope 6300.
6
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell samples were washed with phosphate-buffered saline (PBS) three times and collected; brain tissues were ground 20 to 30 times using Dounce Tissue Grinders (Thermo Fisher Scientific, K8853000002). The samples were lysed in radioimmunoprecipitation assay buffer [50 mM tris (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 0.1% SDS, 0.5% DOC, and 1% Triton X-100] with a protease inhibitor cocktail (Mei5bio, MF182-plus-10), and sonicated six times. The samples were SurePAGE Bis-Tris gels (GenScript, M00652) for electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk at room temperature for 1 hour and incubated with the corresponding primary antibodies in 3% bovine serum albumin (BSA) at 4°C overnight. The next day, the membranes were washed three times in 1× PBS and incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies in 5% skim milk at room temperature for 1 hour. The signals were developed with ECL solution (Millipore, WBKLS0500) after washing three times in 1× PBS. The images were acquired digitally using Clinx ChemiScope 6300.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!