Bestatin

Manufactured by Thermo Fisher Scientific

Sourced in United States

Bestatin is a laboratory reagent used as an aminopeptidase inhibitor. It functions by blocking the enzymatic activity of aminopeptidases, which are enzymes that cleave amino acids from the N-terminus of proteins and peptides.

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Bestatin hydrochloride (2 citations)

5 protocols using bestatin

Brain Tissue Isolation and Protein Extraction

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The 6-month old female mice (C57BL/6) were purchased from Jackson Laboratory (Mouse 1–7). The 18-month old female mice (C57BL/6) (Mouse 8–16) were obtained from the NIH National Institute on Aging (NIA) ageing mouse colony. Mice were euthanized by cervical dislocation, and the brains used for these studies were harvested immediately and kept at −20°C until tissue cell lysates were prepared.
Immediately prior to lysis, organs were cut into small pieces with a straight razor and combined with 400 µL of 20mM phosphate buffer (pH 7.4) and 4 µL of 100X protease inhibitor cocktail in Lysing Matrix A tubes (MP Biomedicals, LLC Solon, OH). The 100X protease inhibitor cocktail included pepstatin A (0.2 mM), leupeptin (0.4 mM), E-64 (0.3 nM), bestatin (1 mM), and AEBSF (20 mM) protease (all from ThermoFisher Scientific, Waltham, MA). The resulting mixture was added to a MP Fast-Prep-24 Tissue Homogenizer in which the sample was homogenized for 20 seconds at 4m/s, put on ice for 5 minutes, and homogenized again for 20 seconds at 4m/s. The homogenized sample was centrifuged for 90 minutes at 4°C and 14,000 rcf. The supernatant was removed and kept at −20°C until it was subject to an iTRAQ-SPROX analysis as described below.

Roberts J.H., Liu F., Karnuta J.M, & Fitzgerald M.C. (2016). Discovery of Age-Related Protein Folding Stability Differences in the Mouse Brain Proteome. Journal of proteome research, 15(12), 4731-4741.

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Protease Activity Detection Assay

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We used either commercially available protease inhibitor peptides or growth of bacterial strains on the protease detection plates. Protease inhibitors were α-2-macroglobulin (7.8 mg/ml in sterile water; Thermo Fisher), aprotinin (0.3 mM in sterile dH₂0; Thermo Fisher), leupeptin (10 mM in sterile dH₂0; Thermo Fisher), and bestatin (1 mM in methanol; Thermo Fisher) that were pipeted as 2.0 μl drops onto the surface of the protein containing plates and allowed to be absorbed into the plate and diffuse for 1 hour. The bacterial strains Photorabdus (Xenorhabdus) luminescens strain Hm primary and secondary forms, Escherichia coli B, Citrobacter freundii, Staphylococcus aureus, S. epidermidis and Enterococcus faecalis were allowed to grow two days at 30°C. Following incubation, the bacterial strains were washed off the plate using a gentle stream of water in order to remove the colonies and eliminate their potential for surface inhibition effects on diffusion of the protease and/or dyes.

Quintero D, & Bermudes D. (2014). A Culture-Based Method for Determining the Production of Secreted Protease Inhibitors. Journal of microbiological methods, 100, 105-110.

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Protein and Phenotypic Analysis of Cell Lines

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CAF, MCF-10A, ZR-75-1, HCT116, QBC939, NCCIT, RBE, HUCCT1, ZJU-0826, and ZJU-1125 cells were washed thrice with ice-cold phosphate buffered saline (PBS), lysed in RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% sodium deoxyholate, 1% Nonide P-40, and 0.1% SDS with Pierce Protease Inhibitor Tablets (AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstain A) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and separated by centrifugation. The protein concentration was detected using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc). Antibodies against the following proteins were used: E-cadherin, N-cadherin, β-catenin, α-SMA, MUC1, CD146, SOX17, Vitamin D3 Receptor (VDR), pdx1, CD326, FoxA1/HNF3α, FoxA2/HNF3β, Nanog, GAPDH, and β-actin (all 1:1000, from Cell Signaling Technology, Danvers, MA, USA).
Flow cytometry was performed using a FACScan instrument (BD Bioscience, San Jose, CA, USA) and commercially available reagents for cells at passages 3–5 and 60–65. A panel of monoclonal antibodies was evaluated, including CD24, CD44, CD29, CD34, CD90, CD117, CD133, CD184, CD326, and CD338 (Biolegend, San Diego, CA, USA). Antigen expression was determined based on a significant shift in staining compared to an isotype control.

Zhang Y., Luo J., Dong X., Yang F., Zhang M., Zhao J., Wang Q., Zhou F., Sun J, & Yang X. (2019). Establishment and Characterization of Two Novel Cholangiocarcinoma Cell Lines. Annals of Surgical Oncology, 26(12), 4134-4147.

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Quantifying Neurochemical Markers in Brain Regions

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To prepare the tissues for the assays 0.3 mL (frontal cortex, striatum) or 0.2 mL (amygdala) of PBS (0.01 mol/L, pH 7.2) containing a protease and phosphatase inhibitor cocktail (aprotinin, bestatin, E-64; leupeptin, NaF, sodium orthovanadate, sodium pyrophosphate, β-glycerophosphate; Thermo Scientific, Rockford, IL) was added to each sample. Each tissue was minced, homogenized, sonicated with an ultrasonic cell disrupter, and centrifuged at 5,000 g at 4°C for 10 min. Supernatants were removed and stored at −20°C until ELISA assays were performed. Bradford protein assays were also performed to determine total protein concentrations in each sample. D1R, D2R (Cat# SEB299Ra and SEA673Ra, Cloud-Clone Corp., Houston, TX) and PSA-NCAM (Cat# 67-ABC0027B, ALPCO Diagnostics, Salem, NH) protein levels were determined using a commercially-available ELISA kits. The assays were performed according to the manufacturer’s instructions. The sensitivity of the assays is 0.055 ng/mL for D1R, 0.112 ng/mL for D2R, and 0.25 ng/mL for PSA-NCAM, and the detection range is 0.156–10 ng/mL for D1R, 0.312–20 ng/mL for D2R, and 0.25–16 ng/mL for PSA-NCAM. The concentration of each protein is presented as ng/mg of total protein accounting for dilution factor.

Stolyarova A, & Izquierdo A. (2015). Distinct patterns of outcome valuation and amygdala-prefrontal cortex synaptic remodeling in adolescence and adulthood. Frontiers in Behavioral Neuroscience, 9, 115.

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Quantitative Proteomic Analysis Protocol

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The following materials were from Sigma Aldrich (St. Louis, MO): glucose, guanidine hydrochloride

(GdmCl)

, S-methylmethanethiosulfonate (MMTS), urea, centrifugal filter units (Amicon Ultra, 0.5 mL, 10

K

MWCO), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), trifluoroacetic acid (TFA), triethylammonium bicarbonate buffer (TEAB, 1 M, pH 8.5), hydrogen peroxide (H₂O₂) (30% w/w), acetic acid, 2-mercaptoethanol, myoglobin from equine heart, proteinase

K

from Tritirachium album, and phenylmethylsulfonyl fluoride (PMSF). The following materials were from ThermoScientific (Waltham, MA): acetonitrile (ACN, LC-MS grade), 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), bestatin, E-64, leupeptin, pepstatin A, TMT10-Plex isobaric label reagent set and porcine pancreas trypsin (proteomics grade). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was from Santa Cruz Biotechnology (Dallas, TX). Phosphate-buffered saline 10X, Molecular Biology Grade (PBS, pH 7.4) was from Corning (Corning, NY). Yeast extract and peptone were from HiMedia (Mumbai, India). Adenine hemisulfate dihydrate was from MP Biomedicals (Santa Ana, CA). Macrospin columns (silica C18) and Pi³ Methionine reagent kit were from Nest Group (Southborough, MA). Research-grade xenon was from Airgas (Randor, PA). Screw top GC vials and caps were from Agilent Technologies (Santa Clara, CA).

Wiebelhaus N., Singh N., Zhang P., Craig S.L., Beratan D.N, & Fitzgerald M.C. (2022). Discovery of the Xenon-Protein Interactome using Large-Scale Measurements of Protein Folding and Stability. Journal of the American Chemical Society, 144(9), 3925-3938.

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