SEC was performed as previously described with modifications17 (link). 10-day-old Col-0 and pag1–2 seedlings were harvested and ground to fine powder in liquid nitrogen and mixed with 2 ml/g of extraction buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 4 mM MgCl2, 200 μM ZnCl2, 0.1% Triton X-100, 1% Glycerol, 4X EDTA-free protease inhibitor (Roche), 2 mM PMSF, and 15 μM MG132). The total protein extracts were centrifuged twice at 4°C for 15 min at 15000 rpm. Then the supernatant was filtered through a 0.2 μm filter. Next, the total protein extracts for each sample was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) that was pre-washed with a balance buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 4 mM MgCl2, 200 μM ZnCl2, 0.1% Triton X-100, 1% Glycerol, 1/3X EDTA-free protease inhibitor (Roche), 0.5 mM PMSF, and 15 μM MG132). The running buffer contained 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 4 mM MgCl2, 200 μM ZnCl2, 0.1% Triton X-100, 1% Glycerol, 1X EDTA-free protease inhibitor (Roche), 2 mM PMSF, and 15 μM MG132. Fractions were collected for western blot analysis using an anti-SE antibody for SE. The Superdex 200 column was also calibrated by gel filtration standard (Bio-Rad).
Edta free protease inhibitor
Manufactured by Roche
Sourced in United States, Switzerland, Germany, China, Canada
EDTA-free protease inhibitor is a laboratory reagent designed to prevent the degradation of proteins by inhibiting protease activity. It is a versatile tool used in various applications, such as protein extraction, purification, and stabilization.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop phosphatase inhibitor, by Roche (11 mentions)
Benzonase, by Merck Group (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (6 mentions)
Nupage gel, by Thermo Fisher Scientific (6 mentions)
Clarity western ecl substrate, by Bio-Rad (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Mg132, by Merck Group (4 mentions)
Chemidoc xrs imaging system, by Bio-Rad (4 mentions)
Anti flag m2 affinity gel, by Merck Group (4 mentions)
Phosstop, by Roche (4 mentions)
Phosphatase inhibitor, by Roche (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dc protein assay kit, by Bio-Rad (4 mentions)
Dnase 1, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Wet transfer apparatus, by Bio-Rad (3 mentions)
Blotting grade blocker non fat dry milk, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Phosphatase inhibitor, by Merck Group (3 mentions)
209 protocols using edta free protease inhibitor
1
Subcellular Fractionation of Arabidopsis Proteins
2
Subcellular Fractionation of Arabidopsis Proteins
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SEC was performed as previously described with modifications17 (link). 10-day-old Col-0 and pag1–2 seedlings were harvested and ground to fine powder in liquid nitrogen and mixed with 2 ml/g of extraction buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 4 mM MgCl2, 200 μM ZnCl2, 0.1% Triton X-100, 1% Glycerol, 4X EDTA-free protease inhibitor (Roche), 2 mM PMSF, and 15 μM MG132). The total protein extracts were centrifuged twice at 4°C for 15 min at 15000 rpm. Then the supernatant was filtered through a 0.2 μm filter. Next, the total protein extracts for each sample was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) that was pre-washed with a balance buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 4 mM MgCl2, 200 μM ZnCl2, 0.1% Triton X-100, 1% Glycerol, 1/3X EDTA-free protease inhibitor (Roche), 0.5 mM PMSF, and 15 μM MG132). The running buffer contained 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 4 mM MgCl2, 200 μM ZnCl2, 0.1% Triton X-100, 1% Glycerol, 1X EDTA-free protease inhibitor (Roche), 2 mM PMSF, and 15 μM MG132. Fractions were collected for western blot analysis using an anti-SE antibody for SE. The Superdex 200 column was also calibrated by gel filtration standard (Bio-Rad).
3
Affinity Purification of EGFP-Tagged Proteins
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EGFP and EGFP–CCHFV-N protein IP experiments were performed using a single-domain anti-GFP antibody conjugated to agarose beads (GFP-trap; Chromotek). IPs with similarly conjugated red fluorescent protein (RFP-trap) were performed as nonbinding negative controls. Cell pellets were incubated for 30 min with 200 μl lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, 1× EDTA-free protease inhibitor [Roche]). The lysate was clarified and diluted 5-fold with dilution buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 1× EDTA-free protease inhibitor [Roche]). The GFP-trap/RFP-trap beads were incubated with diluted cell lysate for 2 h at 4°C and then centrifuged at 2,700 × g for 2 min followed by two washes with wash buffer (10 mM Tris HCl [pH 7.5], 250 mM NaCl, 0.5 mM EDTA, 1× EDTA-free protease inhibitor [Roche]) and then one wash with wash buffer containing 275 mM NaCl. After centrifugation at 2,700 × g, pelleted beads were resuspended in 2× LDS-sample buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 1× protease inhibitor cocktail, 1× LDS, 50 mM dithiothreitol [DTT]) and heated at 95°C for 10 min. Equal volumes of labeled IP samples were mixed and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
4
Nucleo-Cytoplasmic Protein Fractionation
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Cells were harvested by trypsinization, washed with PBS, resuspended in Lysis Buffer (10 mM HEPES pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM MgCl2, 1 mM DTT, 0.2% NP40, 1x EDTA-free protease inhibitor (Roche)) and incubated on ice for 2 min. After centrifugation for 1 min at 800 g, the supernatant containing cytoplasmic proteins was collected in a new tube; the pellet was washed with washing buffer (10 mM HEPES pH 7.9, 60 mM KCl, 1 mM MgCl2, 1 mM DTT, 1x EDTA-free protease inhibitor), centrifuged as previously and then resuspended in nuclear buffer (250 mM Tris-HCl pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM MgCl2, 1 mM DTT, 0.2% NP40, 1x EDTA-free protease inhibitor (Roche), 10% glycerol) and shaken vigorously for 30 min at 4°C. After centrifugation for 30 min at 16 000 g, the supernatant containing nuclear proteins was collected in a new tube. The quality of the nucleo-cytoplasmic separation was assessed by Western blot analysis by detection of β-actin and lamin A/C.
5
Synaptosome Isolation and Lysis
6
GFP-trap co-immunoprecipitation of BRCA1 variants
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GFP-trap co-immunoprecipitation were performed as in ref. 61 (link). In brief, a confluent 15 cm dish of RPE1 TP53−/−BRCA1−/− cells rescued or not with GFP-tagged BRCA1 wild type or I26A mutant were scrapped, washed in ice-cold PBS and lysed in 1 mL of ice-cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.5% Triton X-100, EDTA-free protease inhibitors (Roche) and 20 mM N-Ethylmaleimide (NEM)). Lysates were vortexed and incubated for 1 h at 4 °C while rotating with 500 Units of Benzonase (Millipore). Afterwards, samples were centrifuged for 1 h at 20,000 × g at 4 °C. As inputs, 20 µL of supernatants were saved per sample and the rest was incubated with 25 µL of GFP-Trap beads slurry (Chromotek) for 90 min at 4 °C while rotating. Subsequently, beads were washed three times with wash buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM MgCl2, EDTA-free protease inhibitors (Roche), and 20 mM N-Ethylmaleimide (NEM)) and resuspended in LDS sample buffer 1X for immunoblotting procedures.
7
Synaptosome Isolation and Lysis
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VM or striatal dissections were homogenized in 1 mL of ice-cold 0.32 M sucrose with 5 mM HEPES pH 7.4, 10 mM MgCl2, 100 μg/mL CHX, 13 EDTA-free protease inhibitors (Roche), and 100 U/mL SUPERaseIN. Nuclei and large debris were cleared at 2,000×g for 10 min at 4°C. The supernatant (S1) was further centrifuged at 7,000×g for 15 min at 4°C to yield the P2 pellet. The supernatant (S2) (cytoplasm and light membranes) was removed from the P2 pellet, which was washed by resuspension in 1 mL of ice-cold 0.32 M sucrose buffer (HEPES, MgCl2, CHX, and inhibitors as above) and re-centrifuged at 10,000×g at 4°C before lysis. P2 pellets were lysed in 1 mL of lysis buffer (5 mM HEPES pH 7.4, 150 mM KCl, 10 μm MgCl2, 1% Igepal CA-620, 100 μg/mL CHX, 1 × EDTA-free protease inhibitors [Roche], and 100 U/mL SUPERaseIN). After resuspension, samples were incubated at 4°C on a rotor for 15 min. The resulting synaptosome lysate was subjected to RiboTag IP as described below.
8
Immunoprecipitation of Protein Complexes
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Immunoprecipitation of proteins was done essentially as per published protocols25 (link)50 (link). Cells were lysed in a lysis buffer [20 mM Tris-HCl pH 7.4, 200 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 1 × EDTA-free protease inhibitor (Roche), 5 mM Vanadyl ribonucleoside comples (Sigma), 0.5% Triton X-100, 0.5% sodium deoxycholate] at 4 °C for 15 min, followed by clearing the lysate at 3,000 g for 10 min. Protein G agarose beads (Invitrogen) were blocked with 5% BSA in lysis buffer for 1 h and then incubated with required primary antibody for another 3–4 h before the lyaste was added. A final dilution of 1:50 (antibody:lysate) was used for immunoprecipitation. Immunoprecipitation was carried out for 16 h at 4 °C. Post washing with IP buffer (20 mM Tris-HCl pH 7.4, 150 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 × EDTA-free protease inhibitor (Roche)), the beads were divided in two halves: one subjected to RNA isolation with TRIzol LS and another for western blotting.
9
Co-Immunoprecipitation of Mononucleosome Complexes
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Co-IPs were conducted on mononucleosome fractions derived by Micrococcal Nuclease (MNase) treatment. Nuclei were prepared as above and resuspended in chromatin digestion buffer [50 mM Tris pH 7.4, 4 mM MgCl2, 1 mM CaCl2, EDTA-free protease inhibitor (Roche), 5 mM sodium butyrate] and incubated with MNase (20 KU, NEB) for 15 min at 37°C before the reaction was stopped by adding EDTA to a final concentration of 10 mM. The MNase fraction was derived by centrifugation at 10,000 rpm 5 min at 4°C. The MNase fraction was diluted 1/7 in RIPA buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% IGEPAL CA-630, 1 mM EDTA, EDTA-free protease inhibitor (Roche)] and protein complexes were precipitated over night at 4°C with protein G agarose beads (GE Healthcare) and mouse anti-Ty1 or rat anti-HA antibodies. The complexes were washed five times in RIPA buffer and eluted using 2 × Laemmli buffer (125 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.005% bromophenol blue, 5% beta-mercaptoethanol). Co-IP extracts were analyzed by western blot or processed for Mass Spectrometry.
10
Subcellular Fractionation of Parasites
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Parasites were freed from RBCs by lysis in 0.075% saponin in PBS. The cytoplasmic (CP) fraction was derived by incubating the parasites in lysis buffer [20 mM HEPES pH 7.8, 10 mM KCl, 1 mM EDTA, 1 mM DTT, EDTA-free protease Inhibitor (Roche)] for 30 min on ice, after which IGEPAL CA630 was added to a final concentration of 0.65%. Parasites were centrifuged at 2,500 g for 10 min at 4°C and the cytoplasmic (CP) fraction was stored at -80°C. The pellet containing the nuclei was washed once in lysis buffer and further extracted in nuclear extraction buffer [20 mM HEPES pH 7.8, 800 mM KCl, 1 mM EDTA, 1 mM DTT, EDTA-free protease Inhibitor (Roche)] for 30 min at 4°C, followed by centrifugation at 2,500 g for 30 min at 4°C and the nuclear soluble (Ns) supernatant diluted with the same volume of dilution buffer (20 mM HEPES pH 7.8, 1 mM EDTA, 1 mM DTT, 30% glycerol). The resulting pellet (P) was extracted with 2 × Laemmli buffer (P) and all fractions were analyzed by western blot on 4–12% BisTris gradient gels in 1 × MES running buffer (Invitrogen).
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