Papain from papaya latex

Manufactured by Merck Group

Sourced in Canada, United States, Germany, Italy, Switzerland, United Kingdom

Papain from papaya latex is a proteolytic enzyme that can be used for various laboratory applications. It is derived from the latex of the papaya plant and is known for its ability to break down proteins. The core function of papain is to catalyze the hydrolysis of peptide bonds in proteins.

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24 protocols using papain from papaya latex

Vegan Enzyme Characterization Protocol

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Pepsin from porcine gastric mucosa (lyophilized powder, ≥3200 units/mg protein, lot # SLCH7086), α-Chymo Trypsin from bovine pancreas (lyophilized powder, ≥40 units/mg protein, lot # SLCH1926), Papain from papaya latex (≥8.0 units/mg protein, lot # SLCJ3270) and Trypsin from bovine pancreas (powder, ≥7500 BAEE units/mg solid, lot # SLCM7280) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Here, papain was chosen since it comes from a vegetable to fully meet expectations of vegan or vegetarian people.

Bernier M.È., Thibodeau J, & Bazinet L. (2024). Enzymatic Hydrolysis of Water Lentil (Duckweed): An Emerging Source of Proteins for the Production of Antihypertensive Fractions. Foods, 13(2), 323.

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Isolation of Mouse VLM Neurons

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Mouse VLM neurons were acutely isolated according to our previously described technique42 (link). Briefly, WT, ASIC1 and 2 KO male mice (C57BL/6 genomic background), 8 to 12 weeks of age, were anesthetized with halothane and sacrificed by decapitation using a guillotine. The whole brain was removed and placed in cold extracellular solution (ECF), and the medulla was subsequently sectioned at 250~300 μm with a microtome (Leica VT 1000 S, Germany). The slices were then incubated in ECF containing 3–5 mg/ml papain (from papaya latex, Sigma-Aldrich Chemical Co., USA) at room temperature for 20 to 30 min. All ECFs were bubbled with 100% O₂. Following the enzymatic digestion, slices were washed three times and incubated in enzyme-free ECF for at least 30 min before dissociation. To isolate VLM neurons, individual slices were transferred into a 35 mm culture dishes containing 2 ml of ECF and each dish containing each slice was placed on the stage of an inverted phase-contrast microscope for identifying the VLM region. The VLM region of the slice was excised and single cells were mechanically dissociated using two fire polished glass pipettes or fine forceps. Electrophysiological recording of isolated VLM neurons began approximately 30 min after mechanical dissociation.

Song N., Guan R., Jiang Q., Hassanzadeh C.J., Chu Y., Zhao X., Wang X., Yang D., Du Q., Chu X.P, & Shen L. (2016). Acid-sensing ion channels are expressed in the ventrolateral medulla and contribute to central chemoreception. Scientific Reports, 6, 38777.

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Quantification of Hyaluronic Acid in Decellularized Collagen Matrices

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HA amount was quantified using an established protocol [14 (link)]. Briefly, collagen matrices were decellularized by incubation with distillated water for 1 h. Afterwards, decellularized collagen matrices were digested with digestion solution consisting of papain solution (0.02 mg/mL papain from papaya latex (Sigma-Aldrich), 10 mM EDTA (Sigma-Aldrich), 5 mM L-cysteine (Sigma-Aldrich) in 5× PBS for 2 h at 60 °C. The amount of HA production was quantified using commercial HA-ELISA kit (TECOmedical Group, Sissach, Switzerland) following manufacturer’s protocol.

Sapudom J., Müller C.D., Nguyen K.T., Martin S., Anderegg U, & Pompe T. (2020). Matrix Remodeling and Hyaluronan Production by Myofibroblasts and Cancer-Associated Fibroblasts in 3D Collagen Matrices. Gels, 6(4), 33.

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Quantitative Elemental Analysis of Spheroids

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Spheroids were thawed and enzymatically digested at 65 °C under constant agitation at 500 rpm for 48h in 1 ml of 250 mM phosphate buffer containing 25 mM EDTA (Thermo Fisher) and 1.5 U papain from papaya latex (Sigma-Aldrich). Samples were diluted 1:20 in 1 % HNO₃ (Nitric acid 65 % suprapur, Sigma-Aldrich) and subsequently analyzed in collision/reaction cell mode by inductively coupled plasma mass spectrometry (ICP-MS, ICapQ, Thermo Fisher), using external and internal standard calibration.

Zimmerer A., Schulze F., Gebhardt S., Huesker K., Stobbe D., Grolimund D., Hesse B., Wassilew G.I, & Schoon J. (2024). Impact of gadolinium-based MRI contrast agent and local anesthetics co-administration on chondrogenic gadolinium uptake and cytotoxicity. Heliyon, 10(8), e29719.

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Production and Characterization of Fab Fragments

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2G4 Fab was produced from 2G4 IgG (RSR Ltd) using papain from Papaya latex (Sigma-Aldrich) using an IgG:papain ratio of 50:1 w/w (Horimoto et al. 1992 (link)). Digestion reactions were incubated at 37°C in PBS (8.1 mmol/L Na₂HPO₄, 1.5 mmol/L KH₂PO₄, 2.7 mmol/L KCl, 137 mmol/L NaCl pH 7.4) plus l-cysteine (8.25 mmol/L) and ethylenediaminetetraacetic acid (1.65 mmol/L) for 4 h with occasional mixing by inversion. The reactions were stopped by the addition of iodoacetamide (to 47 mmol/L) and incubation at room temperature for 30 min.
4F5 Fab was produced from 4F5 IgG (RSR Ltd) by papain digestion with 30:1 w/w IgG:papain ratio as described for 2G4 IgG earlier (Hendry 2001a , Hendry et al. 2001b (link)).
Digested 2G4 IgG or 4F5 IgG mixtures were purified on a MabSelect column (Cytiva, Little Chalfont, UK) in 150 mmol/L sodium chloride, 1 mol/L glycine, pH 8.6. The non-binding fractions were collected as the Fab pool and the concentrations were determined from the absorbance at 280 nm using an extinction coefficient of 1.333 mol mg⁻¹ cm⁻¹ as determined by ProtParam.
The 2G4 Fab and 4F5 Fab were analysed by SDS-PAGE (Laemmli 1970 (link)) and by analytical SEC.

Baker S., Miguel R.N., Thomas D., Powell M., Furmaniak J, & Smith B.R. (2022). Cryo-electron microscopy structures of human thyroid peroxidase (TPO) in complex with TPO antibodies. Journal of Molecular Endocrinology, 70(3), e220149.

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Photoconversion of Larval Brains

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For photoconversion experiments, 96 h larval brains were dissected in Chang & Gerhing solution (3.2 g/L NaCl, 3 g/L KCL, 0.69 g/L CaCl₂-₂H2O, 3.7 g/L MgSO₄-7H₂O, 1.79 g/L tricine buffer pH 7, 3.6 g/L glucose, 17.1 g/L sucrose, 1 g/L BSA) at room temperature. Brains were then dissociated in Chang & Gerhing solution supplemented in collagenase from Clostridium histolyticum (Sigma) and papain from papaya latex (Sigma) at a final concentration of 1 mg/mL each, during 30 minutes at 30 °C. Brains were washed with imaging medium (see above) and then dissociated in imaging medium by pipetting 20–30 times.

Roubinet C., Tsankova A., Pham T.T., Monnard A., Caussinus E., Affolter M, & Cabernard C. (2017). Spatio-temporally separated cortical flows and spindle geometry establish physical asymmetry in fly neural stem cells. Nature Communications, 8, 1383.

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Biochemical Reagent Preparation and Use

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Diethylpyrocarbonate (DEPC), imidazole, iodoacetamide, L-cysteine, papain from papaya latex, tris(2-carboxyethyl)phosphine (TCEP), and DL-dithiothreitol (DTT) were obtained from Sigma Aldrich (St. Louis, MO). The mAb immunoglobulin G1 (IgG1) was purchased from Waters Corporation (Milford, MA). Human β-2-microglobulin (β2m) was obtained from Fitzgerald Industries International (Concord, MA). Recombinant Human Growth Hormone (HGH) was purchased from Biovision (San Francisco, CA). Urea was purchased from Acros Organics (Geel, Belgium). Both immobilized trypsin and chymotrypsin were obtained from Princeton Separations (Adelphia, NJ). Sodium phosphate monobasic monohydrate was purchased from EM Science (Darmstadt, Germany). Sodium phosphate dibasic anhydrous, hydrogen peroxide, methanol, formic acid, acetonitrile, and water were purchased from Fisher Scientific (Fair Lawn, NJ). Centricon molecular weight cutoff (MWCO) filters were obtained from Millipore (Burlington, MA).

Borotto N.B., Zhou Y., Hollingsworth S.R., Hale J.E., Graban E.M., Vaughan R.C, & Vachet R.W. (2015). Investigating Therapeutic Protein Structure with Diethylpyro-carbonate Labeling and Mass Spectrometry. Analytical chemistry, 87(20), 10627-10634.

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Papain-induced Lung Inflammation in Mice

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Mice were anesthetized under aerosolized isoflurane and immediately instilled with 10 μg of papain from papaya latex (Sigma, St. Louis, MO) intranasally (i.n.) in 40 μl of sterile PBS on days 18, 19, and 20 post-infection with T. muris. The day after the last instillation of papain, mice were injected intraperitoneally (i.p.) with 2,2,2-tribromoethanol (Avertin; Sigma), tracheas were cannulated, and bronchoalveolar lavages (BALs) were performed using triplicates of 1 ml of sterile 10% fetal bovine serum in PBS. BAL fluid was then red cell lysed using ammonium chloride buffer. Lung tissue was digested in 200 U/ml collagenase type IV (Sigma) for 1 h, red cell lysed, and centrifuged in a 30% Percoll solution to purify leukocytes. BAL fluid and lung cells were analyzed by flow cytometry.

Chenery A.L., Antignano F., Burrows K., Scheer S., Perona-Wright G, & Zaph C. (2016). Low-Dose Intestinal Trichuris muris Infection Alters the Lung Immune Microenvironment and Can Suppress Allergic Airway Inflammation. Infection and Immunity, 84(2), 491-501.

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Quantitative Analysis of Tissue-Derived Biomolecules

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For DNA quantification, each tissue macrospheroid containing 100,000 OACs was digested overnight at 60 °C in 1 mL of papain digestion buffer, containing: 20 µL of papain solution (Papain from papaya latex, Sigma-Aldrich), 8 mg sodium acetate (Sigma-Aldrich), 1.6 mg cysteine hydrochloride (Sigma-Aldrich), 18.6 mg EDTA (Sigma-Aldrich) and up to 1 mL of deionized water. Next, supernatants (100 µL) were transferred in duplicate to clear bottom black 96-well microplates (Costar) and 100 µL of Quanti-iT™ PicoGreen^® ds DNA Assay reagent (Molecular Probes, Eugene, OR, USA) was added into each well. Finally, fluorescence was measured using the TECAN Infinite M200 plate reader (TECAN) at 485 nm excitation and 520 nm emission wavelengths, to determine DNA contents.
The quantities of GAG were determined by using the Sulfated Gycosaminoglycan Assay (Blyscan™ Kit, Biocolor, UK). Briefly, 40 µL of supernatants taken from papain digested samples or same volumes of chondroitin-6 sulphate standards, were transferred to clear polystyrene 96-well plates (Costar). Following the addition of 200 µL of 1.9-dimethylmethylene blue solution (Sigma-Aldrich) per well, absorbances were measured at 540 and 595 nm on the TECAN Infinite M200 plate reader (TECAN), within 5 min. The OHP content was determined following a modified protocol of Stegemann and Stalder (Supplement 2).¹⁸

Žigon-Branc S., Barlič A., Knežević M., Jeras M, & Vunjak-Novakovic G. (2018). Testing the potency of anti-TNF-α and anti-IL-1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor-matched chondrogenically differentiated mesenchymal stem cells. Biotechnology progress, 34(4), 1045-1058.

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Chondrogenic Differentiation of BM-MSCs

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Fibrin and PF hydrogels (1.5 ml) were prepared as described earlier in 12 well plates. BM-MSC were seeded at a density of 20,000 cells/cm² and grown for 7 days in a chondrogenic medium. Cells were harvested with trypsin/EDTA and lysed with 100 μl of papain extraction buffer containing 0.2 M sodium phosphate buffer (pH 6.4), 10 mM EDTA, 0.1 M sodium acetate, 7 mM L-cysteine and 0.12 mg/ml papain from papaya latex (Sigma-Aldrich) at 65 °C for 3 hours. DNA content was determined using 0.2 mg/ml Hoechst 33258 dye (Sigma-Aldrich) and quantified against salmon testes DNA standard. Samples were quantified with a fluorescence plate reader at 360/460 nm. GAG content was measured using dimethyl-methylene blue (DMMB) dye binding method28 (link). Briefly, 50 μl of papain-digested sample were combined with 100 μl DMMB reagent solution (40 mM NaCl, 40 mM glycine, 46 mM DMMB, pH 3.0). The absorbance was determined at 525 nm using shark cartilage chondroitin sulfate C standard (Sigma-Aldrich).

Goldshmid R., Cohen S., Shachaf Y., Kupershmit I., Sarig-Nadir O., Seliktar D, & Wechsler R. (2015). Steric Interference of Adhesion Supports In-Vitro Chondrogenesis of Mesenchymal Stem Cells on Hydrogels for Cartilage Repair. Scientific Reports, 5, 12607.

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