Limited proteolysis of hproBDNF WT and V66M mutant proteins was carried out at 37 °C at a final concentration of 25 μM in 50 mM Sodium Phosphate pH 7.0 by using a panel of three selected proteases from Proti-Ace kits (Hampton Research, Aliso Viejo, CA, USA), i.e., actinase E, thermolysin, and trypsin at a final concentration of 2 μg/mL. The digestions were quenched after 1, 5, 10, 20, 40, and 60 min by the addition of SDS-PAGE sample buffer to aliquots of the reaction mixtures. The samples were analyzed by performing 12% SDS-PAGE and Coomassie staining.
Proti ace kit
Manufactured by Hampton Research
Sourced in United States
The Proti-Ace Kit is a laboratory equipment designed for protein analysis. It provides the necessary components and protocols for researchers to perform protein quantification, purification, and characterization experiments.
Automatically generated - may contain errors
Lab products found in correlation
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Thermo Fisher Scientific (1 mentions)
Sodium phosphate, by Merck Group (1 mentions)
Ammonium hydroxide, by Thermo Fisher Scientific (1 mentions)
Ethylenediaminetetraacetic acid edta, by Thermo Fisher Scientific (1 mentions)
Lead acetate, by Thermo Fisher Scientific (1 mentions)
Imidazole, by Thermo Fisher Scientific (1 mentions)
Ecolite scintillation cocktail, by MP Biomedicals (1 mentions)
2 4 2 hydroxyethyl piperazin 1 yl ethanesulfonic acid hepes, by Merck Group (1 mentions)
14c serine, by PerkinElmer (1 mentions)
Tris 2 carboxyethyl phosphine tcep, by GoldBio (1 mentions)
Tris hydroxymethyl aminomethane tris, by Merck Group (1 mentions)
L serine, by Merck Group (1 mentions)
Trifluoroacetic acid tfa, by Merck Group (1 mentions)
Dl homocysteine, by Merck Group (1 mentions)
Sds nupage gradient gels, by Thermo Fisher Scientific (1 mentions)
Instant blue safestain, by Abcam (1 mentions)
6 protocols using proti ace kit
1
Limited Proteolysis of hproBDNF Variants
2
Limited Proteolysis of mα-DG-Nt and hα-DG-Nt
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mα‐DG‐Nt and hα‐DG‐Nt were subjected to limited proteolysis at 37 °C at a final concentration of 30 μm in 25 mm Tris pH 7.5, 150 mm NaCl buffer. A panel of proteases from Proti‐Ace kits (Hampton Research, Aliso Viejo, CA, USA), that is, bromelain, proteinase K, subtilisin, thermolysin, trypsin, endoproteinase Glu‐C, and clostripain (endoproteinase Arg‐C), were tested at a final concentration of 2 μg·mL−1. The reactions were stopped after 1, 5, 10, 20, 40, and 60 min by adding SDS sample buffer to aliquots of the reaction mixtures. The samples were analyzed by performing 15% SDS/PAGE and Coomassie staining.
3
Trypsin Digestion and EspB Sequencing
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Samples were incubated with trypsin for different length of time at a molar ratio of 1:6 (enzyme:substrate) following the Proti-Ace™ Kit (Hampton Research) recommendation. The reaction was stopped by adding SDS-PAGE loading buffer (63 mM Tris-HCl, 2% SDS, 10% glycerol, 0.1% bromophenol blue) and samples were resolved on a 12% polyacrylamide gel. Bands were transferred from the SDS-PAGE gel to a PVDF membrane and stained with 0.1% Coomassie Brilliant Blue R-250, 40% methanol, and 10% acetic acid until bands were visible. The membrane was then washed with water and dried, and EspB cleavage products were cut out. The first ten amino acids were determined by Edman sequencing at the Plateforme Protéomique PISSARO IRIB at the Université de Rouen.
4
Homocysteine Metabolism Assay Protocol
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Amicon Ultra 15 centrifugal filters, D,L-homocysteine, L-serine, sodium phosphate, trifluoroacetic acid (TFA), tris(hydroxymethyl)aminomethane (Tris), and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) were all purchased from Sigma. Ammonium hydroxide, hydrochloric acid, and sodium dodecyl sulfate were purchased from Fisher. Ethylenediaminetetraacetic acid (EDTA), imidazole, and lead acetate were purchased from Acros Organics. [14C]-serine was from PerkinElmer. Tris (2-carboxyethyl) phosphine (TCEP) was from GoldBio. S-adenosyl-L-methionine (the disulfate tosylate salt) was purchased from BioVision. Trypsin was obtained from the Hampton Research Proti-ACE kit. EcoLite scintillation cocktail was obtained from MP Biomedicals. AG-50W-X8 anion exchange packing material was obtained from Bio-Rad. The Oligos for site directed mutagenesis were purchased from IDT. Luria Bertani (LB) broth was purchased from Research Products International (RPI).
5
Protease Cleavage Experiments of MBP-ERS
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Endoproteinase Glu-C and α-chymotrypsin used for protease cleavage experiments were from Proti-Ace Kit (Hampton Research HR2-429) and were reconstituted following the manufacturer's protocol. The proteases were diluted to 4 μg/ml in 10 mM HEPES, pH 7.5, 500 mM NaCl. To initiate the reactions, MBP-ERS2.5W proteins under storage condition (4 mg/ml in 12.5 mM Tris-HCl pH 7.5, 75 mM NaCl, 0.5 mM DTT, and 40% glycerol) were mixed with an equal volume of 4 μg/ml protease solutions following a 5-min preincubation at 37 °C. The reactions were incubated at 37 °C for various time periods from 15 min to 5 h and were quenched into SDS-PAGE protein loading buffer. Cleaved protein samples (4 μg) were analyzed by SDS-PAGE.
6
Elastase-Mediated Parkin Protein Digestion
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