A confluent 15-cm dish of HEK293 cells propagating aggregated TauP301S-Venus was harvested by snap freezing. The cell pellet was resuspended in 400 μl cold extraction buffer (10 mm Tris, pH 7.5, 2 mm NaV, 50 mm NaF, 50 mm β-glycerophosphate, PhosSTOP phosphatase inhibitor [Roche], cOmplete EDTA-free protease inhibitor mixture [Roche], 100 mm NaCl) and sonicated briefly. Cell debris was removed by centrifugation at 1000 × g, 4 °C, for 1 min. The cleared lysate was centrifuged at 337,000 × g, 4 °C, for 15 min. The resulting pellet was resuspended in 100 μl extraction buffer with 1% (w/v) Sarkosyl, sonicated again, and then incubated for 1 h at 22 °C, 700 rpm, to extract the Sarkosyl-soluble fraction before repeating the centrifugation step. The Sarkosyl-insoluble pellet was resuspended in disaggregation buffer with protease inhibitors (50 mm Hepes-KOH [pH 7.5], 50 mm KCl, 5 mm MgCl2, 2 mm DTT, 2 mm NaV, 50 mm NaF, 50 mm β-glycerophosphate, PhosSTOP phosphatase inhibitor [Roche], cOmplete EDTA-free protease inhibitor mixture [Roche]), briefly sonicated, and centrifuged to remove the remaining detergent. Finally, the pellet was resuspended in disaggregation buffer with protease inhibitors and stored at 4 °C.
Complete edta free protease inhibitor mixture
Manufactured by Roche
Sourced in Switzerland, United States
Complete EDTA-free protease inhibitor mixture is a laboratory product that effectively inhibits a broad spectrum of proteases without the use of EDTA. It is designed to preserve the integrity of protein samples during extraction and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Imperial protein stain, by Thermo Fisher Scientific (2 mentions)
Pierce bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Glutathione sepharose 4 fast flow, by GE Healthcare (2 mentions)
Amicon ultra concentrator, by Merck Group (2 mentions)
Hitrap desalting column, by GE Healthcare (2 mentions)
Odyssey infrared scanner, by LI COR (2 mentions)
α tubulin antibody t9026, by Merck Group (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
3xflag peptide, by Merck Group (1 mentions)
Mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Bis tris sds page, by Thermo Fisher Scientific (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Odyssey clx imaging system, by Bio-Rad (1 mentions)
Odyssey blocking buffer tbs, by LI COR (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Ecl plus reagent, by GE Healthcare (1 mentions)
32 protocols using complete edta free protease inhibitor mixture
1
Extraction and Fractionation of Aggregated Tau Protein
2
Immunoprecipitation and ATG4B Cleavage Assay
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HeLa cell lines grown in 10-cm dishes were transfected with a total of 2.5 μg of plasmid DNA. After 24 h, cells were washed in cold PBS and lysed in ∼1 ml of IP buffer (50 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and cOmpleteTM EDTA-free Protease Inhibitor Mixture, Roche Applied Science). Lysates were cleared by centrifugation at 15,000 × g for 10 min at 4 °C. After 100 μl input sample was collected into a separate tube and boiled in sample buffer, and the remaining cleared lysate was loaded onto anti-FLAG® M2 Affinity Gel (A2220, Sigma) that had previously been equilibrated by washing three times with 1 ml of IP buffer. Approximately 30 μl of packed gel was used per sample, and binding was performed at 4 °C for 3 h on a tube rotator. Gel was washed three times with 1 ml of IP buffer, and bound protein was eluted from the gel by incubating with 3xFLAG peptide (F4799, Sigma) at 150 ng/μl in IP buffer for 30 min at 4 °C with shaking. Eluted sample (∼100 μl) was carefully removed from pelleted gel prior to digestion with recombinant GST–ATG4B and/or boiling in 5× SDS sample buffer to prepare samples for Western blotting.
3
Western Blot Sample Preparation
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Cells were washed in cold D-PBS prior to lysis on ice using lysis buffer (1% NP40, 100 mM Tris pH8, 100 mM NaCl, 10% glycerol, 5 mM EDTA, cOmpleteTM EDTA-free Protease Inhibitor Mixture, Roche Applied Science) supplemented with 20 mM N-ethylmaleimide. Lysates were cleared by centrifugation at 15 000×g at 4°C, and the resulting pellet was discarded. Total protein concentrations were determined using Pierce BCA protein assay kit (Thermo Fisher Scientific), and lysates were diluted to approximately equal concentrations before addition of 4× sample buffer (containing 10% BME) with immediate boiling at 95°C for 10 min. Proteins were separated by 4–12% bis-tris SDS–PAGE (Invitrogen) in 1× MOPS or MES SDS running buffer (Invitrogen) at 100 V, and transferred (1× Transfer Buffer (Invitrogen), 20% methanol) onto Immobilon-FL PVDF membrane (Millipore) for 1.5 h at 100 V. Membranes were blocked in Odyssey blocking buffer TBS (Licor) or 5% non-fat milk or 1% BSA (Sigma, A7906) for 1 h before overnight incubation at 4°C with primary antibodies. Membranes were scanned the following day after 1 h incubation with secondary antibodies using the Odyssey CLx Imaging System or ImageQuant system (Bio-Rad). The following secondary antibodies were used: HRP anti-rabbit (CST, 7074S) and anti-mouse (CST, 7076S) or LI-COR IRDye® 800CW (anti-rabbit) and IRDye 680RD (anti-mouse).
4
Protein Isolation and Western Blot
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Western blots were performed as described in previous paper with modification13 (link),47 (link). Protein was isolated from cells or myotubes using RIPA buffer (Boston Bioproduct BP115 containing 50 mM Tris, 150 mM NaCl, 10% glycerol, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) with 0.5 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM Na3VO4, and 1 mM NaF and complete EDTA-free protease inhibitor mixture (Roche Applied Science). Cell lysates were disrupted mechanically by passing them through 25 G 5/8 needles 10 times and then centrifuged at 10,000 g for 5 min. The supernatant was collected, and protein concentrations were determined with BCA protein Assay Reagents (ThermoFisher Scientific, 23225) followed by measurement with NanoDrop. Protein samples were mixed with 4X Laemmli sample buffer (BIO-RAD # 161-0747) and same amount of protein (10 μg–40µg) was loaded and separated by Mini-PROTEAN® TGX™Precast Gels (4%–20%), Blots were probed with primary antibody and the Horseradish peroxidase conjugated secondary antibodies (antibodies used in western blots could be found in Supplementary table of primers and antibodies). Immunoblots were visualized using ECL plus reagent (GE Life Sciences), and the Fiji program was used to quantify protein abundance.
5
LAMP-1 and LAMP-2 Immunoprecipitation and Immunoblotting
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Cells were harvested and lysed in lysis buffer (1% Triton X, 20 mM Tris-HCl pH 7.0, 5 mM EDTA, 50 mM NaCl) and 1X complete EDTA-free protease inhibitor mixture (Roche, Basel, Switzerland) followed by centrifugation at 15 000 × g for 15 min at 4 °C to remove the cell debri. The supernatants of the cell lysates were then collected and protein concentrations were determined by BCA Protein Assay Kit (Pierce, Rockford, IL, USA). For immunoprecipitation of LAMP-1 and 2, 1 mg of the total lysates were incubated with 2 μg of anti-LAMP-1 or anti-LAMP-2 antibody overnight at 4 °C with gentle rocking followed by 1- h incubation with 50 μl of protein G-Sepharose 4 Fast (GE Healthcare Life Sciences, Buckinghamshire, UK) at 4 °C. The immunoprecipitated samples were then run on 4–12% NuPAGE (Invitrogen) and electrophoretically transferred to a PVDF membrane for immunoblotting. The blots were probed with primary and AP-conjugated secondary antibodies for 1 h each, and immunoreactive bands were visualized using ECF substrate (GE Healthcare Life Sciences). All western blot images were acquired by Typhoon FLA 9500 (GE Healthcare Life Sciences) and intensity of the bands was quantified by ImageQuant 5.2 (GE Healthcare Life Sciences).
6
AMPAR Ubiquitination Assay in Neurons
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The ubiquitination of AMPARs was induced by incubating neurons in artificial cerebrospinal fluid (ACSF; 25 mm HEPES, 120 mm NaCl, 5 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 30 mm d -glucose, pH 7.4) containing 50 μm AMPA (Tocris), 50 μm d -2-amino-5-phosphonovaleric acid (Tocris), and 1 μm tetrodotoxin (Tocris) for 10 min at 37 °C. Neurons were then lysed in warm 1% SDS (in PBS) and diluted in 10 volumes of ice-cold cell lysis buffer (1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 50 mm NaF, 5 mm sodium pyrophosphate in PBS) supplemented with 10 mm N-ethylmaleimide and Complete EDTA-free protease inhibitor mixture (Roche Applied Science). Lysates were centrifuged at 14,000 rpm for 20 min at 4 °C and cleared with protein A- or G-Sepharose beads. Precleared lysates were then incubated with antibodies (anti-ubiquitin, anti-GluA1, or anti-GFP) coupled to protein A- or G-Sepharose overnight at 4 °C followed by four washes with ice-cold lysis buffer and elution in 2× SDS sample buffer. The immunoprecipitated proteins were resolved by SDS-PAGE and probed by Western blot analysis with specific antibodies against GluA1 and ubiquitin.
7
Purification of His-tagged Recombinant Proteins
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Cell pellets were resuspended in Buffer A (50 mm Tris, 200 mm NaCl, pH 7.5, in Milli-Q water) supplemented with complete EDTA-free protease inhibitor mixture (Roche Applied Science), lysozyme, DNase, and RNase (Sigma). The cells were lysed on ice using a Bandelin Sonoplus sonicator with a TT13/F2 tip, set to 30% power, 20 s on, 40 s off for 30 min. Cellular debris was removed by ultracentrifugation using a Ti50.2 rotor in a Beckman Optima LE-80k ultracentrifuge at 40,000 rpm for 1 h at 4 °C. The supernatant was passed through a 0.45-μm filter. The clarified supernatant was applied to a gravity flow nickel-nitrilotriacetic acid-agarose column (Qiagen). The column was washed with three column volumes of Buffer A supplemented with 10 mm imidazole and then three column volumes of Buffer A supplemented with 40 mm imidazole. Protein was eluted in 1-ml fractions using Buffer A supplemented with 250 mm imidazole. Fractions containing the purified protein were buffer-exchanged into Buffer B (20 mm Tris, 100 mm NaCl, pH 7.5, in Milli-Q water) using a 10DG column (Bio-Rad). Protein aliquots were flash-frozen until required.
8
Outer Membrane Isolation from Bacteria
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Cells of overnight cultures were harvested by centrifugation at 10,000 rpm for 10 min at 4°C. The pellets were gently resuspended in a mixture of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 50 mM MgCl2 containing complete EDTA-free protease inhibitor mixture (Roche Applied Science) followed by sonication. Total membranes were collected by ultracentrifugation at 100,000 × g for 1 h at 4°C. The OM was isolated by differential extraction with the same buffer and 1.5% (vol/vol) Triton X-100 and incubated at 4°C overnight. The OM fractions were recovered by centrifugation at 100,000 × g for 1 h at 4° C.
9
Western Blot Analysis of ORF1 Proteins
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Expression assays were carried out essentially as described in the Supporting Information for Cook et al [49 (link)]. HeLa cells, in six-well plates, were transfected with 1μg of pORF1-Flag constructs using 3μL of FuGENE6 (Promega). After 48 h, the cells were washed with PBS, lysed with 50 mM Tris·Cl pH 7.4, 650 mM NaCl, 1 mM EDTA, 1% Triton X-100, cOmplete EDTA-free protease inhibitor mixture (Roche), 100 μM leupeptin, and sonicated in a Bioruptor (Diagneode) and centrifuged at 17,000 × g for 15 min at 4°. Fifty μg samples of supernatant protein were subjected to denaturing gel electrophoresis, transferred to PVDF membranes using iBlot (Invitrogen), blocked with Superblock T20 buffer (Pierce) and incubated overnight at 4° with mouse anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) and rabbit anti-tubulin (Sigma-Aldrich) that had been diluted in Superblock T20 buffer. After rinsing with 1XTBS/0.05% Tween20, the membranes were subjected to three 10 min washes with the same buffer and incubated for 1.5 h at room temperature with mouse and rabbit anti-horse radish peroxidase antibodies in blocking buffer. After four 10 min washes in 1x TBS/0.05% Tween20, followed by three rinses with 1x TBS the membranes were developed with Pierce Pico-west substrate and exposed to film.
10
Purification of Recombinant Protein Sensors
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