Exponentially growing T. cruzi epimastigotes (108) were treated with extraction buffer [0.1% Triton X-100, 100 mM NaCl, 10 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 300 mM sucrose, 50 mM NaF, 1 mM Na3VO4, 0.5 mM PMSF, and EDTA-free complete protease inhibitor cocktail (Roche)] at 4 °C for 10 min. Samples were centrifuged (3,800 g x 2 min), and the supernatants were saved (SF1). The pelleted cells were treated with the same extraction buffer and centrifuged (3,800 g x 2 min), and the supernatants were saved (SF2). The pellets were treated with 500 units of DNase I for 30 min and subsequently centrifuged (3,800 g x 2 min), and the supernatants were saved (DBPs). Samples were resolved by SDS-PAGE and analyzed by western blot.
Free complete protease inhibitor cocktail
Manufactured by Roche
Sourced in Switzerland
The free complete protease inhibitor cocktail is a laboratory reagent designed to inhibit a broad spectrum of proteases. It provides a convenient and effective way to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Kanamycin, by Thermo Fisher Scientific (1 mentions)
Histidine, by Merck Group (1 mentions)
Ampicillin, by Formedium (1 mentions)
Imidazole, by Merck Group (1 mentions)
Agarose, by Thermo Fisher Scientific (1 mentions)
Sypro orange protein gel stain, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Formedium (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Formedium (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Pcr clean up, by Qiagen (1 mentions)
Qiaprep spin miniprep, by Qiagen (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Plasmid midi kit, by Qiagen (1 mentions)
Tricine, by Merck Group (1 mentions)
4 2 hydroxyethyl piperazine 1 ethanesulfonic acid hepes, by Thermo Fisher Scientific (1 mentions)
Basemuncher endonuclease, by Abcam (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
4 protocols using free complete protease inhibitor cocktail
1
Fractionation of T. cruzi Epimastigotes
2
Native Protein Extraction and BN-PAGE
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Human tissues were homogenized in 7 volumes of BN buffer (1% Triton X-100, 12 mM NaCl, 500 mM 6-aminohexanoic acid, 20 mM Bis–Tris, pH 7.0, 2 mM EDTA, 10% glycerol) added with EDTA-free complete protease inhibitor cocktail, (Roche, Basel, Switzerland). After lysis on ice for 1 h, samples were centrifuged at 17,000× g for 30 min at 4 °C. A BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) was used to determine the total protein content of supernatants. The BN-PAGE experiments were performed as previously described [8 (link)] using 60 μg of protein sample previously mixed with 2 μL of loading buffer (5% Coomassie Blue G-250, 750 mM aminocaproic acid) and 10% glycerol by volume. At the end of gel running, proteins were transferred to PVDF membranes (Millipore, Burlington, MA, USA) for immunoblot analysis, as described below.
3
Recombinant Protein Purification Protocol
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All commercially available chemicals were used without further purification. BaseMuncher endonuclease was purchased from AbCam. Ampicillin, dithiothreitol (DTT), isopropyl β-D-1-thiogalactopyranoside (IPTG) and 2-(N-morpholino)ethanesulfonic acid-sodium dodecyl sulfate (MES-SDS) were purchased from Formedium. DH5α chemically competent Escherichia coli, DpnI were purchased from New England Biolabs (NEB). QIAprep Spin Miniprep, PCR clean-up and Plasmid Midi kits were from Qiagen. Ethylenediaminetetraacetic acid (EDTA)-free Complete protease inhibitor cocktail was from Roche. ATP, C43(DE3) and BL21(DE3) chemically competent E. coli, D2O, glycerol, histidine, imidazole, lysozyme, PRPP, potassium chloride, and tricine were purchased from Sigma-Aldrich. Agarose, dNTPSs, kanamycin, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), MgCl2, NaCl, PageRuler Plus Prestained protein ladder, PageRulerTM Plus Prestained protein ladder, and SYPRO orange protein gel stain were from ThermoFisher Scientific. DNA oligonucleotide primers were synthesised by Integrated DNA technologies (IDT).
4
Evaluating Disease Progression in ALS Mouse Models
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The disease courses of the mice were evaluated according to criteria based on changes in body weight [6 (link)]. Disease onset was regarded as the time when each mouse reached its peak weight. The endpoint was defined as the age at which a mouse was unable to right itself within 5 s after being pushed onto its side. The duration of disease was regarded as the period from onset of disease until the endpoint.
The hSOD1G127X/Becn1+/- mice and their littermates were analyzed at three distinct stages of the disease: a presymptomatic stage (150 days), a symptomatic stage (10 % weight loss), and a terminal stage (n = 5 per genotype per disease stage). The hSOD1G93A/Becn1+/- mice and their littermates were examined at a symptomatic stage (10 % weight loss) and a terminal stage (n = 4 per genotype per disease stage). The lumbar half of the spinal cord was immediately dissected, frozen in liquid nitrogen, and stored at −80 °C until use. The spinal cords of mice were extracted in phosphate-buffered saline (pH 7.0) containing 1 % (v/v) Nonidet P-40 and EDTA-free Complete® protease inhibitor cocktail (Roche Applied Science) as described previously [51 (link)], yielding detergent-soluble and insoluble fractions.
The hSOD1G127X/Becn1+/- mice and their littermates were analyzed at three distinct stages of the disease: a presymptomatic stage (150 days), a symptomatic stage (10 % weight loss), and a terminal stage (n = 5 per genotype per disease stage). The hSOD1G93A/Becn1+/- mice and their littermates were examined at a symptomatic stage (10 % weight loss) and a terminal stage (n = 4 per genotype per disease stage). The lumbar half of the spinal cord was immediately dissected, frozen in liquid nitrogen, and stored at −80 °C until use. The spinal cords of mice were extracted in phosphate-buffered saline (pH 7.0) containing 1 % (v/v) Nonidet P-40 and EDTA-free Complete® protease inhibitor cocktail (Roche Applied Science) as described previously [51 (link)], yielding detergent-soluble and insoluble fractions.
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