Immunoglobulin G and M, transferrin, haptoglobin, alpha-2-macroglobulin, alpha-1-antitrypsin, complement C3, and alpha-1-acid glycoprotein purified from human serum were purchased from Sigma-Aldrich (St. Louis, MO). Immunoglobulin A purified from human plasma was purchased from CalBiochem (La Jolla, CA). Anti-human-IgA (α-chain specific)-agarose and anti-human-IgM (μ-chain specific)-agarose used for protein enrichment were purchased from Sigma-Aldrich (St. Louis, MO). Sequencing grade modified trypsin and dithiothreitol (DTT) were purchased from Promega (Madison, WI). Pronase E from Streptomyces griseus and iodoacetamide (IAA) were purchased from Sigma-Aldrich (St. Louis, MO).
Pronase e from streptomyces griseus
Manufactured by Merck Group
Sourced in United States
Pronase E is a proteolytic enzyme derived from the bacterium Streptomyces griseus. It is a broad-spectrum enzyme that can cleave a wide range of peptide bonds in proteins and polypeptides.
Automatically generated - may contain errors
Lab products found in correlation
Haptoglobin, by Merck Group (1 mentions)
Alpha 2 macroglobulin, by Merck Group (1 mentions)
Alpha 1 antitrypsin, by Merck Group (1 mentions)
Complement c3, by Merck Group (1 mentions)
Alpha 1 acid glycoprotein, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Promega (1 mentions)
Iodoacetamide iaa, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Jupiter c18, by Phenomenex (1 mentions)
Sodium periodate, by Merck Group (1 mentions)
Afb1 standard, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Coumarin, by Merck Group (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Hplc grade methanol, by Avantor (1 mentions)
Ethyl acetate, by Daejung Chemicals (1 mentions)
Complete proteinase inhibitor cocktail, by Roche (1 mentions)
7 protocols using pronase e from streptomyces griseus
1
Purification and Characterization of Human Serum Proteins
2
Protease-Mediated Analysis of Amyloid Fibrils
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Proteinase K: a 180 µl aliquot from a stock solution of seeded or unseeded in vitro fibrils or of ex vivo fibrils (0.2 mg/ml each) was mixed with 20 µl 200 mM Tris buffer, pH 8.0, containing 1.4 M NaCl, 20 mM CaCl2 and 0.4 µl Proteinase K (20 mg/ml, Fermentas). The mixture was incubated at 37 °C and 20 µl aliquots were taken after 0, 10, 30, 60 and 120 min. Proteinase K activity was stopped by adding 0.5 µL of 200 mM PMSF solution that was dissolved in methanol. After 10 min of incubation the samples was frozen in liquid nitrogen until they were analyzed. The proteolytic digestion products were analyzed using denaturing protein gel electrophoresis.
Pronase E: a 90 µl aliquot from a stock solution of seeded and unseeded in vitro fibrils (0.2 mg/ml each) was mixed with 5 µl 2 M Tris buffer, pH 7.5 and 5 µl pronase E from Streptomyces griseus (0.8 mg/ml, Sigma-Aldrich). The samples were incubated at 37 °C and 15 µl aliquots were taken after 0, 10, 30, 60 and 120 min. The pronase E activity was stopped by adding 2.25 µl cOmplete EDTA-free protease inhibitor cocktail (Roche) that was prepared by dissolving 1 tablet in 7 ml Millipore water. After 10 min of incubation at room temperature, the samples were plunge frozen in liquid nitrogen until they were analyzed using denaturing protein gel electrophoresis.
Pronase E: a 90 µl aliquot from a stock solution of seeded and unseeded in vitro fibrils (0.2 mg/ml each) was mixed with 5 µl 2 M Tris buffer, pH 7.5 and 5 µl pronase E from Streptomyces griseus (0.8 mg/ml, Sigma-Aldrich). The samples were incubated at 37 °C and 15 µl aliquots were taken after 0, 10, 30, 60 and 120 min. The pronase E activity was stopped by adding 2.25 µl cOmplete EDTA-free protease inhibitor cocktail (Roche) that was prepared by dissolving 1 tablet in 7 ml Millipore water. After 10 min of incubation at room temperature, the samples were plunge frozen in liquid nitrogen until they were analyzed using denaturing protein gel electrophoresis.
3
Pronase E Digestion Kinetics of Peptides
4
Aflatoxin B1 Detection Protocol
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AFB1 standard, RBBR, coumarin, sodium periodate, and trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Pronase E from Streptomyces griseus was also purchased from Sigma-Aldrich. HPLC grade methanol and acetonitrile were obtained from J.T. Baker (Avantor Performance Materials, Inc., Center Valley, PA, USA). Ethyl acetate was purchased from Daejung Chemicals and Metals Co. (Gyeonggi-do, Korea).
5
Melanoidins Isolation and Fractionation
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The melanoidins were isolated from the macromolecular fractions obtained after dialysis following the protocol of Morales and van Boekel [11 (link)], but including enzymatic hydrolysis in the process. In a typical run, the I and S fractions were suspended in 50 mM phosphate buffer pH 7.0 (20 mg/mL) and treated with 1% Pronase E from Streptomyces griseus (4 UI/mg, Sigma-Aldrich, St. Louis, MI, USA) at 37 °C for 24 h with magnetic agitation, and then centrifuged at 4,500 rpm for 30 min at 4 °C. The supernatants obtained from the treatment of the I and S fractions were separated and labeled IHS and SH, respectively, and fractionated by SEC-HPLC on a Biogel P2 column irrigated with aqueous acetic acid (0.2% v/v). This process was repeated thrice.
6
Enzymatic Extraction of Macromolecular Fractions
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The I and S macromolecular fractions were suspended in 50 mM phosphate buffer at pH 7.0 to a final concentration of 20 mg/mL and treated with 40 UI/mL of Pronase E from Streptomyces griseus (4 UI/mg, Sigma-Aldrich, St. Louis, MI, USA) at 37°C for 24 h with magnetic stirring, and then centrifuged at 4,500 rpm for 30 min at 4°C. The supernatants obtained from the treatment of the I and S fractions were separated by centrifugation, freeze-dried, and labeled IHS and SH, respectively (Figure 1 ) (14 (link)).
7
Protease Treatment of Infected Cells
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Infected mid- and late-state IEs (24–36 hours post invasion) were washed twice in PBS and incubated at 5% hematocrit with 1 mg/mL Pronase E from Streptomyces griseus (Sigma) in PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2 for 30 min at 37 °C or 1 μg/mL of porcine-modified trypsin (Sigma) in PBS for 30 min at 37 °C. After extensive washing with AB buffer supplemented with complete Proteinase Inhibitor Cocktail (Roche, Basel, Switzerland), the treated cells were used for aptamer-binding assays.
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