Frozen liver tissue was homogenized in 50 mM HEPES, 150 mM NaCl, 10% Glycerol, 0.1% Tween 20, 7.5 mM EDTA, and 7.5 mM MgCl2*6H2O. Protein content was determined using a DC™ Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA), and homogenates were diluted to 5 mg/ml. To reduce nonspecific binding of the anti-collagen type I antibody, liver homogenates were digested with pepsin prior to electrophoresis. To accomplish this, pepsin (Powder 1:3000; VWR, Radnor, PA) was diluted to 2 mg/ml in 2 N HCl, and 10 μl of this pepsin preparation was added to 100 μl of liver homogenate (500 μg protein). This sample was incubated for 2 hours at 20°C and then neutralized with 10 μl of 2 N NaOH and resuspended in SDS loading buffer (100 mM Tris-Cl pH 6.8, 4% SDS, 0.2% bromophenol blue and 20% glycerol) containing 400 mM β-mercaptoethanol. pepsin-digested samples (6 μl/lane) were resolved on an 8% SDS-polyacrylamide gel, transferred to nitrocellulose, and incubated with an anti-collagen type I antibody (#AB765P, EMD Millipore, Hayward, CA). Undigested liver homogenates (25 μg protein/lane) were probed with an anti-actin antibody (Santa Cruz Biotech, Dallas, TX) and served as a quasi-loading control. Blots were subsequently incubated with species-specific, HRP-conjugated secondary antibodies, and bands were visualized with Pierce™ ECL Western Blotting Substrate (Thermo Scientific).
Pepsin
Manufactured by Avantor
Sourced in United States
Pepsin is a proteolytic enzyme derived from the gastric mucosa of animals, primarily pigs. Its core function is to catalyze the hydrolysis of proteins into smaller peptides, contributing to the digestion process. Pepsin is commonly used in various laboratory applications, such as protein analysis and preparation.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Avantor (3 mentions)
Elisa, by PerkinElmer (1 mentions)
Api 4000, by AB Sciex (1 mentions)
1200 series, by Agilent Technologies (1 mentions)
Avance 400, by Bruker (1 mentions)
Potassium dihydrogen phosphate, by Sinopharm (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Solarbio (1 mentions)
Disodium hydrogen phosphate, by Sinopharm (1 mentions)
Acyclovir, by Meilun (1 mentions)
Sodium dihydrogen phosphate, by Sinopharm (1 mentions)
Bile salt, by Merck Group (1 mentions)
Pancreatin, by Merck Group (1 mentions)
Gel extraction kit, by Takara Bio (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sangon (1 mentions)
Mouse anti his monoclonal antibody, by Tiangen Biotech (1 mentions)
Tryptone and yeast extract, by Thermo Fisher Scientific (1 mentions)
Dna marker, by Takara Bio (1 mentions)
Protease k, by Avantor (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
2 2 azobis 2 amidinopropane dihydrochloride, by Merck Group (1 mentions)
11 protocols using pepsin
1
Collagen Type I Detection in Liver Tissue
2
Cytarabine Nanoparticle Development and Evaluation
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Cytarabine (Ara-C) was purchased from the Aladdin Industrial Corporation. Palmitic acid (PA), sodium hydroxide, potassium dihydrogen phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate were bought from Sinopharm Chemical Reagent Co., Ltd. Ethyl chloroformate (EtOCOCl) was obtained from Chengdu Beisite Reagent Co., Ltd. Pepsin (1 : 3000) and trypsin (1 : 250) were purchased from Amresco and Sangon Biotech (Shanghai) Co., Ltd, respectively. Acyclovir (HPLC > 98%) was purchased from Dalian Meilun Biotech Co., Ltd. HL60 and K562 cells were kindly provided by the Immunopharmacology Institute of Shandong University and the Shandong Analysis and Test Center. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Beijing Solarbio Technology Co., Ltd.
The spectroscopy equipment was: an NMR spectrometer (1H-NMR, Bruker Avance 400), an electrospray tandem mass spectrometer (MS, AB SCIEX API 4000), an FTIR spectrometer (Nicolet 6700), a transmission electron microscope (TEM, JEM-200CX), a high performance liquid chromatograph (HPLC, Agilent Technologies 1200 Series), an HPLC-MS (Agilent 1260 triple quadrupole mass spectrometer equipped with an ESI source), and a microplate reader (ELISA, PerkinElmer).
The spectroscopy equipment was: an NMR spectrometer (1H-NMR, Bruker Avance 400), an electrospray tandem mass spectrometer (MS, AB SCIEX API 4000), an FTIR spectrometer (Nicolet 6700), a transmission electron microscope (TEM, JEM-200CX), a high performance liquid chromatograph (HPLC, Agilent Technologies 1200 Series), an HPLC-MS (Agilent 1260 triple quadrupole mass spectrometer equipped with an ESI source), and a microplate reader (ELISA, PerkinElmer).
3
Evaluating Probiotic Survivability in Simulated Digestive Conditions
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To evaluate the survivability of L. lactis in simulated gastric condition, one piece of each enteric coated capsule with L. lactis film, non-enteric coated capsule with L. lactis film, L. lactis film and 1 ml of free bacteria cells were digested in 10 ml filtered SGF containing 2000 U/ml pepsin (Amresco, USA) in sterile PBS at pH 2. Samples were digested in separate tubes at 37 °C and harvested every hour up to 4 hours for survivability evaluation. Digested films, capsules and free bacterial cells were then centrifuged at 3900 rpm for 5 minutes at 4 °C and washed with PBS. Samples were serially diluted with PBS and plated on M17 agar supplemented with 0.5% glucose (w/v) and 10 μg/ml chloramphenicol followed by incubation at 30 °C for 48 hours for viable cell count (CFU/ml). The entire processes was repeated in filtered SIF containing 3mg/ml pancreatin (Sigma Aldrich, USA) and 0.1% (w/v) bile salt (Sigma Aldrich, USA) at pH 8. Sequential digestion was done by digesting samples in SGF for 2 hours followed by SIF for 4 hours. Films, capsules and free bacterial cells without digestion were used as normalization controls. The survivability (%) was determined using the equation below:
4
Phage Stability in Digestive Fluids
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The stability of the wilt type and mutant phages in gastric and pancreatic enzymatic conditions was assessed. For this, simulated gastric fluid (SGF) comprised of 3.2 mg.mL−1 pepsin (Amresco) in 0.2% (wt/vol) NaCl at pH 3.5, and artificial pancreatic fluid (APF) comprised of 1 mg.mL−1 pancreatin (Biocatalysts) in 0.2% (wt/vol) NaCl at pH 8.0 were prepared. Phages were added to the pre-warmed solutions at 37 °C at a concentration of 108 pfus.mL−1 and incubated at 37 °C. Samples were taken at 0, 5, 30, 60, 90 and 120 min and immediately 10-fold serially diluted in SM buffer to determine phage titre. Controls with the same conditions but without the enzymes were used. The experiments were repeated twice and the results presented as mean log survival ± standard deviation of the phages in the enzymatic fluids compared to the respective controls.
5
Recombinant Protein Expression in E. coli
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The strains and vectors used in this study are shown in Table S1 . E. coli Top10 stored in our laboratory was used for all cloning steps. For recombinant proteins expression, pET-21a vector (Novagen, Madison, WI) was used with expression host E. coli BL21(DE3)pLysS (Novagen, Madison, WI). Restriction enzymes, Pyrobest DNA Ploymerase, dNTP, DNA Marker and gel extraction kits were obtained from TaKaRa (Dalian, China). The mouse anti-His monoclonal antibody and enhanced horseradish peroxidase (HRP)-Diaminobenzidine (DAB) chromogenic substrate kit were obtained from Tiangen (Beijing, China). FITC labeled donkey anti-mouse IgG, HRP labeled rabbit anti-mouse IgG, lead acetate, IPTG, and antibiotics were all purchased from Sangon Biotech (Shanghai, China). Pepsin (1:3000) was obtained from Amresco (OH, USA). Tryptone and yeast extract were obtained from Oxoid (Basingstoke, UK). E. coli was cultured in Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract and 1% NaCl) supplemented with antimicrobial agents, as necessary.
6
Synthesis and Characterization of α-Butyl Cyanoacrylate Adhesive
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An α-butyl cyanoacrylate (BCA) monomer was purchased from Beijing Suncon Medical Adhesive Co., Ltd. (Beijing, China). Poloxamer 188 (BASF) was obtained from Beijing Fengli Jingqiu Trade Limited Liability Co., Ltd. (Beijing, China). CTS was purchased from Zhenjiang Aoxing Biological Technology Co., Ltd. (Zhenjiang, China). The CG was synthesized by our laboratory. Dextran-70 (BR) was purchased from Shanghai Crystal Pure Reagent Co., Ltd. (Shanghai, China). TP5 was purchased from Shanghai Soho-Yiming Pharmaceuticals Co., Ltd (Shanghai, China). Pepsin (1:3,000, USP grade) and trypsin (1:250, >1,000 N.F.U/ mG, EC 3.4.21.4, Amresco) were obtained from Shanghai Technical Service Industry and Biological Engineering Co., Ltd. (Shanghai, China). All other chemicals used in this study were of analytical reagent grade without further purification. Deionized water was used for the preparation of all solutions.
Male Sprague Dawley rats (190±10 g) were provided by The Experimental Animal Center of Zhejiang Province (Hangzhou, China). All animal experiments were approved by the Animal Ethical Committee of Jiangsu University and complied with the Guidelines for Care and Use of Laboratory Animals.
Male Sprague Dawley rats (190±10 g) were provided by The Experimental Animal Center of Zhejiang Province (Hangzhou, China). All animal experiments were approved by the Animal Ethical Committee of Jiangsu University and complied with the Guidelines for Care and Use of Laboratory Animals.
7
Characterizing Antifungal Properties of rF2
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To analyze the effect of different pHs (range 3.0–10.0) on the antifungal activity of rF2, the pH of the culture filtrate was altered using 2 mol/l NaOH or HCl and incubated for 2 h at 37 °C. Protein rF2 was exposed to a range of temperatures (40, 60, 80, 100, and 121 °C) for 30 min to study its thermal stability. To detect its stability under protease treatment, rF2 was digested using 1 mg/ml protease K, trypsin, and pepsin (Amresco, Radnor, PA, USA) for 60 min at 37 °C, respectively. The antifungal activities of the rF2 culture filtrates treated as detailed above were determined according to the method described by Zhao et al. [37 (link)]. The formula reported by Wong et al. was used to calculate the relative activity of the treated rF2 protein [38 (link)]. Three replicates for each treatment were assessed in three repeated experiments.
8
Xuanwei Ham Antioxidant Capacity Analysis
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Xuanwei hams were provided by Xuanwei Haopin Ham Company (Xuanwei, Yunnan, China). 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), ethylenediaminetetraacetic acid (EDTA), 1,1′-diphenyl-2-picrylhydrazyl (DPPH), fluorescein sodium salt, 2,2′-azobis-2-amidinopropane dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), ferrozine, ACE (from rabbit lung) and N-hippuryl-his-leu hydrate (HHL) were purchased from Sigma Aldrich, Co. (St Louis, MO, USA). The antioxidant capacity assay kit for the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method was purchased from Beyotime Biotechnology (Shanghai, China). Pepsin was obtained from Amresco (Solon, OH, USA). Trypsin was obtained from Gibco (Carlsbad, CA, USA). All other chemicals and reagents used were of analytical grade and obtained in China.
9
Clofazimine Solubility Enhancement Protocol
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Clofazimine (CAS registry number 2030-63-9)
was purchased from Beijing Mesochem Technology Co., Ltd. Coformers,
including hydrochloric acid (37%, Sigma-Aldrich), sulfuric acid (95–97%,
Sigma-Aldrich), nitric acid (70%, Sigma-Aldrich), oxalic acid anhydrous
(>99%, Sigma-Aldrich), ortho-phosphoric acid (85%,
Sigma-Aldrich), citric acid monohydrate (>99%, Sigma-Aldrich),
formic
acid (acetic acid (>95%, Sigma-Aldrich)), and acetic acid (99.8%,
Sigma-Aldrich) were used as received. Methanol (>99%) being of
HPLC
grade and used without further purification was also purchased from
Sigma-Aldrich. Ingredients for the dissolution media; pepsin (extracted
from porcine mucosa, Amresco), sodium taurocholate hydrate (NaTc,
>97%, Sigma-Aldrich), L-α-phosphatidylcholine (lecithin,
∼
99% purity, from bovine brain, Sigma-Aldrich), sodium chloride (Fisher
Scientific), maleic acid (>99%, Sigma-Aldrich), and hydrochloric
acid
(37%, Sigma-Aldrich) were used as received from suppliers. Brain Heart
Infusion Broth (Sigma-Aldrich) was used as a medium for bioactivity
assays.
was purchased from Beijing Mesochem Technology Co., Ltd. Coformers,
including hydrochloric acid (37%, Sigma-Aldrich), sulfuric acid (95–97%,
Sigma-Aldrich), nitric acid (70%, Sigma-Aldrich), oxalic acid anhydrous
(>99%, Sigma-Aldrich), ortho-phosphoric acid (85%,
Sigma-Aldrich), citric acid monohydrate (>99%, Sigma-Aldrich),
formic
acid (acetic acid (>95%, Sigma-Aldrich)), and acetic acid (99.8%,
Sigma-Aldrich) were used as received. Methanol (>99%) being of
HPLC
grade and used without further purification was also purchased from
Sigma-Aldrich. Ingredients for the dissolution media; pepsin (extracted
from porcine mucosa, Amresco), sodium taurocholate hydrate (NaTc,
>97%, Sigma-Aldrich), L-α-phosphatidylcholine (lecithin,
∼
99% purity, from bovine brain, Sigma-Aldrich), sodium chloride (Fisher
Scientific), maleic acid (>99%, Sigma-Aldrich), and hydrochloric
acid
(37%, Sigma-Aldrich) were used as received from suppliers. Brain Heart
Infusion Broth (Sigma-Aldrich) was used as a medium for bioactivity
assays.
10
Cardiotoxin Degradation in Gastric Juice
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After deprivation of food and water for 24 h, the ICR male mice (number: 2007000564768, SLAC, Shanghai) were ligated pylorus under anesthesia. Then the mice were administrated cardiotoxin at dose of 20 mg/kg body weight for 15 minutes. The gastric juice (GJ) was extracted out and the supernatant was separated for assay of cardiotoxin after centrifugation at 12,000 rpm for 10 minutes at 4°C. For in vitro assay, cardiotoxin was incubated in artificial gastric juice (AGJ) containing 1% pepsin (0685, AMRESCO, LLC) and 1.64% dilute hydrochloric acid (pH 1–1.2) or in distilled water with pH adjusted to 1.2 with HCl for 15 minutes. After centrifugation at 12,000 rpm for 10 minutes at 4°C, the supernatant was separated for degradation assay. The supernatants were loaded into PAGEL and separated by electrophoresis and dyed with imperial protein stain (24615, Thermo Scientific).
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