Leupeptin

Manufactured by Fujifilm

Sourced in Japan

Leupeptin is a laboratory reagent used as a protease inhibitor. It functions by inhibiting the activity of certain enzymes, particularly serine and cysteine proteases. Leupeptin is commonly used in research applications to prevent the degradation of proteins during sample preparation and analysis.

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Lab products found in correlation

Aprotinin, by Fujifilm (3 mentions) Pd123319, by Fujifilm (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) Dynabeads protein g, by Thermo Fisher Scientific (1 mentions) Antarctic phosphatase, by New England Biolabs (1 mentions) T4 pnk, by New England Biolabs (1 mentions) Human serum albumin (hsa), by Merck Group (1 mentions) Lps escherichia coli o55 b5, by Merck Group (1 mentions) Nafamostat mesylate, by Tokyo Chemical Industry (1 mentions) Iodoacetamide, by Fujifilm (1 mentions) Calcein, by Dojindo Laboratories (1 mentions) β cyclodextrin, by Merck Group (1 mentions) Kod plus neo dna polymerase, by Toyobo (1 mentions) Revertaid m mulv reverse transcriptase, by Thermo Fisher Scientific (1 mentions) N 6 biotinamido hexyl 3 2 pyridyldithio propionamide hpdp biotin, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Streptavidin agarose, by Thermo Fisher Scientific (1 mentions) N ethylmaleimide, by Merck Group (1 mentions) Dithiothreitol (dtt), by Fujifilm (1 mentions) Anti bip antibody, by GeneTex (1 mentions)

8 protocols using leupeptin

Azilsartan and NHE3 Regulation in Proximal Tubule Cells

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Opossum kidney (OK) proximal tubular cells (American Type Culture Collection, Manassas, VA, USA) were cultured at 37°C in an atmosphere of 95% air/5% CO₂ in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). Once confluent, the OK cells were treated with azilsartan (1.0 × 10⁻⁶ mol/l), candesartan (3.0 × 10⁻⁷ mol/l), or vehicle for 24 h, and simultaneously with angiotensin II (1.0 × 10⁻¹¹ mol/l).
To evaluate the contributions of different protein-degradation routes, the effect of azilsartan on NHE3 was examined in the presence of inhibitors of lysosomal (leupeptin 0.5 μg/ml) (Wako) or proteasomal (lactacystin 10 μmol/l) (Wako) pathways of degradation. To assess the involvement of the angiotensin II type-2 receptor (AT2R), the AT2R antagonist PD123319 (10 μmol/l; (Wako) was used.

Hatanaka M., Kaimori J.Y., Yamamoto S., Matsui I., Hamano T., Takabatake Y., Ecelbarger C.M., Takahara S., Isaka Y, & Rakugi H. (2016). Azilsartan Improves Salt Sensitivity by Modulating the Proximal Tubular Na+-H+ Exchanger-3 in Mice. PLoS ONE, 11(1), e0147786.

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Immunoprecipitation and Small RNA Isolation

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Immunoprecipitation and small RNA isolation were performed as described previously^{48 (link)}. In brief, OSCs or BmN4 cells ware lysed in buffer (30 mm HEPES-KOH (pH 7.4), 500 mm NaCl, 150 mm KOAc, 5 mm Mg(OAc)₂, 5 mm DTT, 0.1% NP-40, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 0.5% aprotinin (Wako)), and then centrifuged. The supernatants were incubated with Dynabeads Protein G (Thermo Fisher Scientific) bound to an anti-DDDDK-tag mAb (MBL, FLA-1). The beads were washed twice with the buffer and twice with the buffer without 500 mm NaCl, and then treated with Proteinase K and phenol-chloroform. The liberated RNAs were precipitated with ethanol, dephosphorylated with Antarctic Phosphatase (NEB), and then radiolabeled with ³²P-γ-ATP (PerkinElmer) and T4 PNK (NEB).

Yamaguchi S., Oe A., Nishida K.M., Yamashita K., Kajiya A., Hirano S., Matsumoto N., Dohmae N., Ishitani R., Saito K., Siomi H., Nishimasu H., Siomi M.C, & Nureki O. (2020). Crystal structure of Drosophila Piwi. Nature Communications, 11, 858.

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Inflammatory Mediator Quantification Protocol

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Human serum albumin (HSA), phosphate-buffered saline (PBS), cerulein and LPS (Escherichia coli O55:B5) were purchased from Sigma-Aldrich (St Louis, Mo). Rat-derived antimouse Gr-1 fluorescein isothiocyanate was purchased from eBioscience (San Diego, Calif). Rabbit-derived antihuman HRG polyclonal antibody was created in our laboratory. Goat-derived antirabbit IgG antibody was purchased from MBL (Nagoya, Japan). d-Phenylalanyl-arginyl chloromethyl ketone was purchased from Cayman Chemical (Ann Arbor, Mich). Aprotinin, leupeptin, and benzylsulfonyl fluoride was purchased from Wako Pure Chemical Industries (Osaka, Japan). Nafamostat mesylate was purchased from Tokyo Chemical Industry (Tokyo, Japan). Benzamidine hydrochloride was purchased from nacalai tesque (Kyoto, Japan).

Terao K., Wake H., Adachi N., Liu K., Teshigawara K., Takahashi H., Mori S, & Nishibori M. (2018). Histidine-Rich Glycoprotein Suppresses Hyperinflammatory Responses of Lung in a Severe Acute Pancreatitis Mouse Model. Pancreas, 47(9), 1156-1164.

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Proteinase Inhibitors for Protein Analysis

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The proteinase inhibitors Nα-p-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) and leupeptin were purchased from Wako (Osaka, Japan) and Peptide Institute (Osaka, Japan), respectively. Iodoacetamide was purchased from Wako (Osaka, Japan).

Yukitake H., Shoji M., Sato K., Handa Y., Naito M., Imada K, & Nakayama K. (2020). PorA, a conserved C-terminal domain-containing protein, impacts the PorXY-SigP signaling of the type IX secretion system. Scientific Reports, 10, 21109.

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Streptozotocin-Induced Diabetes Model with Adjuvants

Cited 2 times

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Streptozotocin (SIGMA) was diluted in citrate sodium buffer, and administered intraperitoneally at the dose of 150 mg/kg of body weight (BW) twice with an interval of 4 days^{26 (link)}. Mice were used for experiments 4 weeks after the treatments. Leupeptin (Wako) was diluted in phosphate-buffered saline, and administered intraperitoneally at the dose of 40 mg/kg of BW 4 h before sacrifice^{56 (link)}. Calcein (Dojindo) was diluted in NaHCO3 solution, and administered subcutaneously at the dose of 8 mg/kg of BW twice, 8 days and 2 days before sacrifice^{57 (link)}. AS1842856, or 5-amino-7-(cyclohexylamino)−1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Universal Biologicals), was diluted in 6% β-cyclodextrin (Sigma), and administered orally at the dose of 100 mg/kg of BW^{32 (link)} once daily for 4 weeks.

Sasako T., Umehara T., Soeda K., Kaneko K., Suzuki M., Kobayashi N., Okazaki Y., Tamura-Nakano M., Chiba T., Accili D., Kahn C.R., Noda T., Asahara H., Yamauchi T., Kadowaki T, & Ueki K. (2022). Deletion of skeletal muscle Akt1/2 causes osteosarcopenia and reduces lifespan in mice. Nature Communications, 13, 5655.

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Cysteine-based redox regulation assay

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DTT, dipyridyl, NH₄Cl, an anti-DYKDDDDK (FLAG) tag antibody, an anti-Myc tag antibody, leupeptin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Diamide, N-ethylmaleimide (NEM), methoxypolyethylene glycol (PEG) maleimide, fetal bovine serum (FBS), penicillin-streptomycin solution, and S-methyl methanethiosulfonate (MMTS) were obtained from Sigma-Aldrich (St. Louis, MO). (N-[6-(biotinamido)hexyl]-3’-(2’-pyridyldithio) propionamide (Biotin-HPDP) and streptavidin-agarose were purchased from Pierce Biotechnology (Rockford IL). An anti-HSC70 antibody was purchased from Novus Biologicals (Littleton, CO). An anti-BiP antibody was purchased from GeneTex, Inc. (Irvine, CA). Dimethyloxaloylglycine (DMOG), Diamide, and a horseradish peroxidase (HRP) Maleimide Conjugate were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). KOD plus neo DNA polymerase was purchased from TOYOBO (Tokyo, Japan). Revert AidTM M-MuLV Reverse transcriptase was purchased from MBI Fermentas (Vilnius, Lithuania).

Kobayashi Y., Oguro A., Hirata Y, & Imaoka S. (2021). The regulation of Hypoxia-Inducible Factor-1 (HIF-1alpha) expression by Protein Disulfide Isomerase (PDI). PLoS ONE, 16(2), e0246531.

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Affinity Purification of Protein Complexes

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HEK293 cells expressing target protein were washed with ice-cold PBS and lysed with cell lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1% Triton X-100) or radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100) supplemented with 10 μg/ml leupeptin (334-40414; Wako Pure Chemical Industries), 2 μg/ml aprotinin (10236624001; Roche), and 50 μM amidinobenzylsulfonyl fluoride (015-26333; Wako Pure Chemical Industries). Clarified lysates were incubated with anti-DYKDDDDK tag antibody beads (018-22783; Wako Pure Chemical Industries) overnight. Beads were washed with the lysis buffer, and bound proteins were dissolved in SDS sample buffer.
His-scarlet-α-catenin VH1 and GST-tricellulin Ctail was expressed in Escherichia coli (BL21star pRARE) and purified using TALON metal affinity resin (Takara Bio) or Glutathione Sepharose 4B (GE Healthcare). After measuring the concentration of purified proteins, scarlet α-catenin VH1 was loaded on TALON metal affinity resin, and purified GST-tagged tricellulin Ctail was added in cell lysis buffer. After reacting at 4°C for 2 h, the beads were washed with cell lysis buffer, and bound proteins were eluted with elution buffer (50 mM Hepes NaOH, pH 7.5, 100 mM NaCl, and 200 mM imidazole).

Cho Y., Haraguchi D., Shigetomi K., Matsuzawa K., Uchida S, & Ikenouchi J. (2022). Tricellulin secures the epithelial barrier at tricellular junctions by interacting with actomyosin. The Journal of Cell Biology, 221(4), e202009037.

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Western Blotting of B16F10 Cells

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B16F10 cells were seeded in a 6-cm dish at 4 × 10⁵ cells/dish and cultured with α-mg. The cells were washed twice with RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% Na-deoxycholate, 1 mM EDTA, 10 mM NaF, 1 mM Na₃VO₄, 0.8 μM Aprotinin [Wako], 50 μM Bestatin [Wako], 20 μM Leupeptin [Wako], 10 μM Pepstatin A [Wako] and 1 mM AEBSF [Wako]). Then, cells were disrupted using an ultrasonic homogenizer and centrifuged to obtain a supernatant for western blotting. After the antibody reaction, the membrane was treated with LuminoGLO reagent (Cell Signaling Technology) to cause the band of the target protein to emit light, and the band was detected and photographed using AE-9300H Ez-Capture MG (ATTO Corporation, Tokyo, Japan). Each protein detection was performed at least 3 times replication.

Zhou S., Yotsumoto H., Tian Y, & Sakamoto K. (2021). α-Mangostin suppressed melanogenesis in B16F10 murine melanoma cells through GSK3β and ERK signaling pathway. Biochemistry and Biophysics Reports, 26, 100949.

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