We isolated mitochondria from a single rat brain using the protocol of Rosenthal et al. [33 (link)], which was slightly modified to obtain better characteristics [34 (link)]. Rat liver mitochondria were prepared according to Steinbrecht and Kunz [35 (link)] with the following modifications. The liver of one 50–60 days old Wistar rat (approx. 5 g) was immediately transferred into ice-cold solution A (0.3 M sucrose, 3 mM EGTA, pH 7.4) and shaken to wash out blood. Then we minced the liver, added around 30 mL of solution A, and homogenized it at 600 units/s using a potter homogenizer (Potter S, B. Braun Melsungen, Melsungen, Germany). Thereafter, the homogenate was centrifuged at 900× g for 5 min. The supernatant was passed through a cheesecloth and centrifuged at 12,000× g for 10 min. The resulting pellet was dissolved with approx. 20 mL of ice-cold solution B (0.3 sucrose, pH 7.4 adjusted with small amounts of Tris-base). The solution was transferred to a small glass homogenizer and homogenized 10–12 times manually. Finally, the suspension was centrifuged at 12,000× g for 10 min, and the resulting pellet was dissolved in the proportion 160 µL solution B per 1 g liver wet weight.
Potter s
Manufactured by B. Braun
Sourced in Germany
The Potter S is a laboratory homogenizer designed for the disruption and homogenization of biological samples. It features a motorized pestle that operates in a glass or PTFE tissue grinder vessel to efficiently break down and mix various materials.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Victor multilabel plate reader, by PerkinElmer (1 mentions)
Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Gs 15r, by Beckman Coulter (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
10 protocols using potter s
1
Isolation of Rat Brain and Liver Mitochondria
2
Myeloperoxidase Activity Assay in Ileum
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The levels of myeloperoxidase (MPO) activity in the segment of ileum were determined as an indicator of neutrophil accumalation. Tissues were homogenized in 50 mM phosphate buffer (pH 7.4) (1/10, w/v) containing protease inhibitor, 0.2 μM phenylmethanesulphonyl fluoride (PMSF) and 1 mM Ethylenediamin tetra acetic acid (EDTA), at 4°C for 30 s using a homogenizer (Potter S, B. Braun, Germany). Then, appropriate volume of homogenate was used MPO determination.
The method of Suzuki et al. was used with a slight modification [13 (link)]. This method is based on the oxidation of the synthetic substrate 3,3’,5,5’-tetramethyl benzidine (TMB) by MPO. The standard reaction mixture consisted of 500 μl detergent-containing buffer (160 mM potassium phosphate buffer, pH 5.4, 1% hexadecyltrimethylammonium bromide), 100 μl TMB (16 mM, dissolved in dimethylformamid), 50 μl homogenate and 300 μl water. The reaction was started by the addition of 50 μl H2O2 (diluted 0.003%) at 37°C. The rate of MPO-catalyzed oxidation of TMB was followed by recording the increase in absorbance at 655 nm. Considering the initial and linear phase of the reaction, we measured the absorbance change per minute, and one enzyme unit was defined as the amount of enzyme producing one absorbance change per minute under assay conditions. Enzyme activity was calculated as units per gram of wet weight tissue.
The method of Suzuki et al. was used with a slight modification [13 (link)]. This method is based on the oxidation of the synthetic substrate 3,3’,5,5’-tetramethyl benzidine (TMB) by MPO. The standard reaction mixture consisted of 500 μl detergent-containing buffer (160 mM potassium phosphate buffer, pH 5.4, 1% hexadecyltrimethylammonium bromide), 100 μl TMB (16 mM, dissolved in dimethylformamid), 50 μl homogenate and 300 μl water. The reaction was started by the addition of 50 μl H2O2 (diluted 0.003%) at 37°C. The rate of MPO-catalyzed oxidation of TMB was followed by recording the increase in absorbance at 655 nm. Considering the initial and linear phase of the reaction, we measured the absorbance change per minute, and one enzyme unit was defined as the amount of enzyme producing one absorbance change per minute under assay conditions. Enzyme activity was calculated as units per gram of wet weight tissue.
3
Subcellular Fractionation of SERT Cells
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Tetracycline induced T-REx-SERT cells were mechanically disrupted with a hypotonic Tris-buffer (50 mM Tris, pH 7.9, proteinase inhibitor) in a two step procedure: dounce-homogenisation (Potter S, B.Braun, Melsungen, Germany) followed by an ultrasound treatment (sonifier W-250, Branson, CT, USA). Homogenates were centrifuged first at 800 rcf (10’, 4°C), followed by a second centrifugation step at 100,000 rcf (60’, 4°C). The resulting pellet was re-suspended in reaction-buffer RB1 (50 mM Tris-HCl, 150 mM NaCl, 5 mM KCl, proteinase inhibitor, pH 7.9), and stored at -80°C after protein determination (Pierce®BCA Protein Assay, Thermo Scientific, IL, USA).
4
Acetylcholinesterase Activity Quantification
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Brain homogenates were prepared by weighing the hemispheres and adding a 10-fold volume of isotonic HEPES-sucrose buffer (HEPES sodium salt 10mM, Sucrose 0.32 M, pH 7.4). Homogenization was performed with a tissue grinder (Potter S, B. Braun, Melsungen, Germany) at 1100 rpm and 15 strokes. The homogenates were assessed for AChE activity using the Ellman method (20) with minor modifications (21, 22) . Briefly, 50 ml of tissue homogenate containing a final volume of 0.5% Triton X-100 were centrifuged for 10 min at 12,000 g and 4 °C. 10 µl of the supernatant was mixed with Ellman buffer and iso-OMPA (final concentration 100 µM). Acetylthiocholine and dithionitrobenzoic acid (1 mM and 500 µM final concentrations, respectively) were added before measuring the absorbance at 405 nm using a Victor multilabel plate reader (Perkin Elmer, Bedford, USA). Enzyme activity was calculated using a standard curve prepared with each assay and expressed in relation to protein amount (mU/mg protein). Protein determination was carried out using the Bradford method.
5
Affinity Isolation of Endogenous PKA Complexes
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Endogenous PKA complexes were affinity isolated from the colon cancer cell lines SW480, SW620, KM12, SKCO1, SNU175, osteosarcoma cell line U2OS, melanoma cell line A375, and human glioblastoma biopsies. Cells were grown in the appropriate media supplemented with 10% (vol/vol) FBS until they reached high cell density. Cells and tissue were homogenized (lysis buffer: 150 mM NaCl, 50 mM Tris-HCL pH7.4, 1 mM EDTA, 1% Triton, supplemented with standard protease inhibitors and phosphatase inhibitors) using a Potter S (B. Braun Biotech International) with 20 strikes. Following centrifugation (15,000×g, 30 min) we affinity-isolated PKA complexes using Rp-8-AHA-cAMP agarose resin (Biolog, #A012). Next, the proteomic composition of isolated PKA complexes were analyzed with LC-MS/MS on a LTQ-Orbitrap XL mass spectrometer (Thermo Fischer Scientific) coupled to a nano-flow LC system (Eksigent) and the data processing has been performed28 (link).
6
Isolation of Mitochondria from Liver Tissue
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Mitochondria were isolated as described by Fernández-Vizarra and colleagues (2006)70 with slight modifications. Frozen liver (500 mg) was disrupted in homogenization medium (0.075 M sucrose; 0.225 M mannitol; 1 mM EGTA; 0.01% BSA; adjusted to pH 7.4) using a loosely-fitting potter homogenizer (Potter S, B. Braun Biotech International) at 1500 rpm with 20 strokes up and down. The homogenate was centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was transferred into a fresh tube and centrifuged at 12000 × g for 6 min at 4 °C. The resulting pellet was washed in homogenization medium and re-centrifuged at 12000 × g for 6 min at 4 °C. This pellet containing the mitochondrial fraction was solubilized in MAITE buffer (25 mM sucrose; 75 mM sorbitol; 100 mM KCl; 0.05 mM EDTA; 5 mM MgCl2; 10 mM Tris-HCl; 10 mM H3PO4; adjusted to pH 7.4) and centrifuged once more at 12000 × g for 6 min at 4 °C. The final pellet was resuspended in 500 ml MAITE buffer. All steps above were performed on ice. Mitochondrial protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Rockford, USA) with bovine serum albumin as standard. The mitochondria were subsequently snap frozen in liquid nitrogen and stored at −80 °C until further use. All chemicals were purchased from Sigma Aldrich, Steinheim, Germany.
7
Liver Extraction and Homogenization Protocol
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Livers were extracted from mice, following transcardial whole-body perfusion with ice-cold PBS, were preserved by snap-freezing in liquid nitrogen, and were stored at −80°C. For homogenization, 500 μl of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA; 89901) with 100× Halt™ Protease Inhibitor Cocktail (1:100, Thermo Fisher Scientific, 78429) was used per 10 mg of snap-frozen liver tissue. Liver tissue was allowed to sit in RIPA buffer for 10 min at 4°C and was then disrupted by mechanical homogenization for 3 min, at 1,500 rpm and 4°C (POTTER S, B. Braun, 8533032). Samples were centrifuged for 15 min, at 14,000 rpm and 4°C (Beckman Coulter, GS-15R). Supernatants were aliquoted and frozen at −80°C.
8
Isolation of Endogenous PKA Complexes
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Endogenous PKA protein complexes from SW620/SW480/HEK293 cells were affinity-purified as described before [37 (link)]. In short, PKA complexes were isolated under standard conditions without stimulation for baseline binding. Cells were homogenized using a Potter S (B. Braun Biotech International) with 15 strikes (standard lysis buffer: 10 mM sodium phosphate pH 7.2, 150 mM NaCl, 0.5% Triton X-100 supplemented with standard protease inhibitors [PI] and phosphatase inhibitors [PPI]). Cell lysates were clarified (13,000 rpm, 15 min) and endogenous PKA-associated protein complexes were precipitated with PKA-selective Rp-8-AHA-cAMP agarose resin (Biolog, #A012) for 2 h at 4 °C. As negative control experiment, we added excess of cAMP (1 mM) to the lysates to mask the cAMP binding sites in the PKA regulatory subunits for precipitation. Resin-associated proteins were washed four times with standard lysis buffer and eluted with 1% SDS. Finally, resin-associated proteins were subjected to SDS/PAGE followed by immunoblotting, or mass-spectrometry(MS) analysis.
9
Postmortem Brain Tissue Homogenization
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For human postmortem brain samples, 0.6 g of tissue was homogenized in 34 mL extraction buffer (0.5% n-Dodecyl β-D-maltoside, 150 mM NaCl, 50 mM Hepes, pH 7.4) with a glass homogenizer (PotterS from B. Braun) set at 900 rpm for 12 strokes. The homogenate was centrifuged twice at 16,000g for 10 min. All subsequent steps were the same as those for iNs.
10
Fractionation of Cerebellar Proteins
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Cerebella were homogenized in lysis buffer (50 mM Hepes pH 7.4, 100 mM NaCl, 3% Brij 58, 5 mM EDTA, 10 mM Na4P2O7, protease inhibitors). The lysate was mixed with 1.5 ml ice-cold sucrose (final concentration: 40%) and transferred to a Potter S (B.Braun, Germany). A homogenization step was performed and 3 ml of homogenate were transferred into a centrifuge tube. The sample was overlaid with 6 ml ice-cold 30% sucrose and then again overlaid with 3 ml ice-cold 5% sucrose and subjected to centrifugation for 20 h at 200,000 × g @4 °C. Twelve samples of 1 ml obtained and proteins of each sample were precipitated with acetone. Fraction 13 (pellet) was diluted in lysis buffer. Pellets from all fractions were lyophilized and resuspended in SDS protein sample buffer for SDS-PAGE and immunoblotting.
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