Pepstatin

Manufactured by Thermo Fisher Scientific

Pepstatin is a pentapeptide inhibitor of aspartic proteases. It is used in research applications to prevent protein degradation by aspartic proteases such as pepsin, renin, and certain cathepsins.

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Close spelling variants of this product

Pepstatin A (19 citations) Bodipy-FL-Pepstatin A (11 citations) BODIPY-pepstatin-FL (2 citations)

5 protocols using pepstatin

Purification of MBP-5-HT3A-ICD Fusion Protein

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The MBP-5-HT_3A-ICD fusion protein was overexpressed in Escherichia coli (E. coli), BL21-CodonPlus^® (DE3)-RIPL cells (Agilent Technologies; Santa Clara, CA). For large scale purification cells were harvested by centrifugation (4,600 × g for 15 min at 4 °C) and resuspended in lysis buffer (Buffer A; 20 mM Tris, 200 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4) containing a freshly prepared cocktail of protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF); Research Products International Corp., 10 μg/mL leupeptin; Sigma-Aldrich, 7 μg/mL pepstatin; Fisher Scientific), and lysozyme (1 mg/mL; Sigma Aldrich) and DNaseI (20 μg/mL; Sigma-Aldrich). The cell suspension was passed through an EmulsiFlex-C3 high-pressure homogenizer (AVESTIN, Inc., Ottawa, ON, Canada) to achieve cell lysis. Debris was removed by centrifugation (10,000 × g for 40 min at 4 °C) and the lysate was subsequently clarified by a second round of centrifugation for 1 h at 90,000 × g and 4 °C. The resultant supernatant was loaded onto a gravity-packed amylose-resin column (New England BioLabs, Inc.). Unbound proteins were washed extensively with buffer A, and the bound fusion protein was eluted with buffer A containing 20 mM maltose.

Pandhare A., Grozdanov P.N, & Jansen M. (2016). Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors. Scientific Reports, 6, 23921.

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Affinity Purification of CASK Complexes

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HEK293 cells maintained in a 100mm dish were transfected using the calcium phosphate method with 10ug EGFP-N1, GFP-CASK-WT or GFP-CASK^L209P DNA per well. Media was changed 6 hrs post-transfection, and cells were incubated at 37°C, 5% CO₂ for 48 hr. All the following steps were performed at 4°C or on ice. Cells were harvested in PBS and solubilized in RIPA buffer containing PBS, 1% Triton X-100, 0.5% sodium deoxycholate, 2mM EDTA, 1mM EGTA and protease inhibitors (0.1 mg/ml aprotinin, 0.1 mg/ml leupeptin, 0.1 mg/ml pepstatin, and 0.01 mg/ml PMSF; all purchased from Fisher Scientific). Cell lysates were incubated on a rocker for 30min. Supernatants were collected by centrifuging the lysates at 15000rpm for 30min. Frozen rat brain was thawed and homogenized in RIPA buffer and incubated on rocker for 30min. Cell and brain lysates were mixed and incubated overnight in cold room on rocker. GFP-trap™ beads (75 μl) were added to the pre-cleared (incubated with plain agarose beads) cell/brain mixture for each condition and incubated for 1.5 hr at 4°C. The beads were washed three times with PBS containing 1% Triton-X 100. 75ul of 2× SDS sample buffer was added to the beads and boiled (100°C) for 10min. Protein samples were separated using SDS-PAGE and immunoblotted for Mint1, Velis and CASK as described below.

LaConte L.E., Chavan V., DeLuca S., Rubin K., Malc J., Berry S., Summers C.G, & Mukherjee K. (2018). An N-terminal heterozygous missense CASK mutation is associated with microcephaly and bilateral retinal dystrophy plus optic nerve atrophy. American journal of medical genetics. Part A, 179(1), 94-103.

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HEK-293 Cell Transfection and CASK Pulldown

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Human embryonic kidney (HEK-293) cells (ATCC) were plated in 6-well plates (JetBiofil) and maintained in DMEM (Hyclone) containing 10% fetal bovine serum (Hyclone) supplemented with 5 mg/ml penicillin-streptomycin (Hyclone). Cells at 80% confluency were transfected with 10 µg of EGFP-N1, GFP-CASK-WT or GFP-CASK^L209P DNA per well using the calcium phosphate method. Two days later, cells were harvested and solubilized in PBS containing 1% Triton X-100, 2mM EDTA, 2mM DTT and protease inhibitors (0.1 mg/ml aprotinin, 0.1 mg/ml leupeptin, 0.1 mg/ml pepstatin, and 0.01 mg/ml PMSF; all purchased from Fisher Scientific). The lysates were incubated by rocking at 4°C for 30min and then centrifuged for 30 min. at 15,000 rpm at 4°C to collect the supernatants. The pre-cleared supernatants were incubated with 30ul of GST or NxCT-GST beads and incubated on a rocker for 2hr at 4°C. The beads were washed three times with PBS containing 1% Triton X 100 buffer. 30 μl of 2× SDS sample buffer was added to the beads and sample was boiled at 100°C for 10min. Protein samples were separated using SDS-PAGE and immunoblotted for CASK as described below.

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Western Blot Analysis of Sperm Proteins

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Percoll‐washed sperm cells were sonicated and sperm heads and tails were isolated as described previously.
²³ (link) Whole sperm cells, sperm heads and tails were lysed in radio immune preciptation assay buffer (Thermo Scientific) with freshly added protease inhibitors: aprotinin, leupeptin, pepstatin, and PMSF (Thermo Scientific) on ice for 30 min followed by a 15 min spin at 14,000 x g, 4°C. Sperm lysates or CRISP2 precipitates were denatured in 4x sodium dodecyl sulfate (SDS) sample buffer (200 mM Tris‐HCl, pH 6.8, 10% β‐mercapto‐ethanol, 8% SDS, 0.08% bromophenol blue, 40% glycerol) and boiled for 10 min. Samples were loaded onto SDS‐PAGE gels (5% stacking gel, 12% resolving gel) and were blotted onto 0.2 µm nitrocellulose membranes (GE Healthcare) at 100 V for 1 h. After blocking for 3 h at RT in 5% (w/v) BSA in PBS with 0.05% (v/v) Tween‐20 (PBST), membranes were incubated with primary antibodies (Supplementary Table S1) overnight at 4°C. After three washes in PBST for 15 min, membranes were incubated with horse radish peroxidase conjugated secondary antibodies (Table S1) for 1 h at RT. After rinsing four times in PBST for 20 min, membranes were developed using chemiluminescence (enzyme chemilumenescence [ECL]‐detection kit; Supersignal West Pico, Pierce). Migration levels of proteins were visualized using a pre‐stained protein ladder, 10 to 250 kDa (Thermo Scientific).

Zhang M., Chiozzi R.Z., Bromfield E.G., Heck A.J., Helms J.B, & Gadella B.M. (2023). Characterization of acrosin and acrosin binding protein as novel CRISP2 interacting proteins in boar spermatozoa. Andrology, 11(7), 1460-1471.

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Detyrosination Activity Stimulation Assay

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HEK-2KO cells were harvested and lysed in 80 mM Pipes (pH 6.8), 5 mM MgCl₂, and 0.2% NP-40. The suspension was rotated for 30 min at 4°C before centrifugation at 15,000g for 10 min. The cleared supernatant was incubated at 37°C for 2 hours (detyrosination activity stimulation). For some experiments, cell extracts were supplemented with either protease inhibitors or metal divalent cation salts (ZnCl₂, MnCl₂, and CoCl₂ at 2 mM). The different inhibitors and working concentrations used throughout the study were the following: pepstatin (Thermo Fisher Scientific, 2 μM), phenylmethylsulfonyl fluoride (Sigma-Aldrich, 1 mM), leupeptin (Sigma-Aldrich, 50 μM), iodoacetamide (Sigma-Aldrich, 10 μM), N-ethylmaleimide (Sigma-Aldrich, 20 mM), E-64 (Thermo Fisher Scientific, 50 μM), Epoxide-Y (EpoY) (Sigma-Aldrich, 50 μM), EDTA (Sigma-Aldrich, 1 mM), EGTA (Sigma-Aldrich, 1 mM), 1,10-phenanthroline (Sigma-Aldrich, 1 mM), dl-benzylsuccinic acid (BSA) (Sigma-Aldrich, 1 mM), 2-(phosphonomethyl)-pentanedioic acid (Sigma-Aldrich, 1 mM) and 2-guanidinoethylmercaptosuccinic acid (Abcam, 1 mM).

Nicot S., Gillard G., Impheng H., Joachimiak E., Urbach S., Mochizuki K., Wloga D., Juge F, & Rogowski K. (2023). A family of carboxypeptidases catalyzing α- and β-tubulin tail processing and deglutamylation. Science Advances, 9(37), eadi7838.

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