Human fibronectin was purchased from BD Biosciences (Heidelberg, Germany). DAPI, chloroquine, carrageenan, dextran sulfate, lipopolysaccharide (E. coli), filipin III and methyl-β-cyclodextrin from Sigma-Aldrich. MG132 was from Merck, Leupeptin and pepstatin from Applichem. Poly-I:C high molecular weight, Poly-I:C-rhodamine and lipoteichoic acid (S. aureus) were from InvivoGen (San Diego, USA). Phenylmethylsulfonyl fluoride (PMSF) was from Applichem.
Leupeptin
Manufactured by AppliChem
Sourced in Germany
Leupeptin is a protease inhibitor used in biochemical research. It primarily inhibits serine and cysteine proteases, including trypsin, plasmin, and papain. Leupeptin is often used to prevent protein degradation in cell and tissue extracts during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pepstatin, by AppliChem (3 mentions)
Dextran sulfate, by Merck Group (1 mentions)
Carrageenan, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Filipin 3, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by AppliChem (1 mentions)
High molecular weight poly 1 c, by InvivoGen (1 mentions)
Poly 1 c rhodamine, by InvivoGen (1 mentions)
Methyl β cyclodextrin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Human fibronectin, by BD (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by AppliChem (1 mentions)
Na3vo4, by Merck Group (1 mentions)
Sodium azide, by AppliChem (1 mentions)
Thiamin, by Merck Group (1 mentions)
Imidazole, by Merck Group (1 mentions)
Na2hpo4, by Avantor (1 mentions)
Mgcl2, by Avantor (1 mentions)
5 protocols using leupeptin
1
Procurement of Diverse Reagents for Cell Biology
2
CagA Translocation Assay in AGS Cells
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Translocation of CagA into AGS cells was analyzed by co-culturing H. pylori strains with AGS cells and detecting tyrosine phosphorylation of CagA, as previously described [42 (link)]. Briefly, H. pylori and AGS human gastric cells were co-cultured at a MOI of 100:1 for 4 h at 37°C in six-well plates. After infection time the cells were put on ice and washed with PBS. Then PBS containing protease and phosphatase inhibitors (1mM Na3VO4 (Sigma), 1mM PMSF (AppliChem), 1μM leupeptin (AppliChem), 1μM pepstatin (AppliChem)) was added and the cells were scraped off the six-well plates. After centrifugation the cell sediment was resuspended in PBS containing protease and phosphatase inhibitors. CagA translocation was assessed by separating the soluble fraction using SDS-PAGE and immunoblotting with an anti-phosphotyrosine antibody (PY99, Santa Cruz Biotechnology).
3
Isolation of Nanovesicles from Tomato Fruits
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Nanovesicles were isolated from Piccadilly variety tomato fruits (200 g, Vittoria Colonna s.r.l., Sicily, Italy) by differential ultracentrifugation (dUC) according to Bokka et al. [1 (link)]. In brief, after washing and removing exocarp, tomatoes were homogenized in a kitchen mixer homogenizer by 3 cycles of 10 s in extraction buffer composed of 100 mM phosphate, 10 mM ethylenediaminetetraacetic acid (EDTA) (J.T. Baker, Deventer, The Netherlands) (pH 8, 170 mL) containing protease inhibitors 1 mg/mL leupeptin (0.085 mL, AppliChem, Darmstadt, Germany), 100 mM phenylmethylsulfonyl fluoride (PMSF, 0.425 mL) and 1 M sodium azide (0.272 mL) AppliChem, Darmstadt, Germany). The isolation of NVs were performed using four low-velocity centrifugation steps at increasing centrifugal force, i.e., 400× g, 800× g, 2000× g and 15,000× g each of them for 30 min at 22 °C. Supernatant from the last low-velocity centrifugation step was centrifuged at 100,000× g for 2 h at 4 °C using an SW28Ti rotor in Beckman Coulter Optima L-90K ultracentrifuge (Beckman Coulter, Brea, CA, USA). The pellet containing the NVs was re-suspended in a small volume of buffer.
4
Unnatural Amino Acid Incorporation Protocol
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The unnatural amino acid para‐azidophenylalanine was purchased from Bachem GmbH (Bubendorf, Germany). Isopropyl β‐d‐1‐thiogalactopyranoside (IPTG), KCl, leupeptin, pepstatin A and KH2PO4 were purchased from AppliChem (Darmstadt, Germany), MgCl2 was purchased from VWR (Leuven, Belgium), NaCl, thiamin and imidazole from Sigma‐Aldrich (Schnelldorf, Germany), NaOH and Na2HPO4 from VWR Chemicals (Darmstadt, Germany) and FeCl3 was purchased from Merck (Darmstadt, Germany). Standard media ingredients, EDTA and SDS were purchased from Roth (Karlsruhe, Germany).
5
Protein Extraction and Western Blot Analysis
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Cells were lysed in buffer containing 10 mmol/l Tris/HCl, pH 8.0, 150 mmol/l NaCl, 10 mmol/l, 5 mmol/l EDTA, 0.5 % Triton X-100, 60 mmol/l N-octylglucoside, supplemented with 40 mg/l PMSF and protease inhibitor mix (antipain, aprotinin, leupeptin, chymostatin, pepstatin, trypsin inhibitor; 2 mg/ml each; all from AppliChem). After determination of protein concentration by Bradford assay, equal amounts of proteins were boiled in Laemmli buffer and separated by SDS-PAGE gel. After transfer of proteins onto nitrocellulose membranes and blocking with Roti-Block (Carl Roth), the following primary antibodies were used: anti-flotillin-1, flotillin-2 both from BD Biosciences (610821, 610384) and Santa Cruz (sc-25506, sc-25507). Caveolin-1 and caveolin-2 from Abnova (MAB2408) and Cell Signaling (8522). The antibody for VCAM-1(sc-8304), ICAM-1(sc-8439) and p65(sc-109) were from Santa Cruz, GAPDH (G8795) from Sigma-Aldrich. IRF-3 (4302) and pIRF-3 (Ser 385) (4947) were from Cell Signaling. PERK (Thr 981) (sc-32577) and eIF2α (Ser52) (sc-101670) from Santa Cruz. Western blot analysis was performed with an infrared-based laser scanning detection system (Odyssey, Licor). The infrared fluorescent-dye-conjugated secondary antibodies were purchased from Licor.
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