Glu c

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Glu-C is a lab equipment product from Thermo Fisher Scientific. It is a proteolytic enzyme that specifically cleaves peptide bonds on the C-terminal side of glutamic acid residues in polypeptides.

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Endoproteinase Glu-C (3 citations)

7 protocols using glu c

Ni-NTA Protein Purification and Digestion

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Ni-NTA purified proteins were transferred onto Vivacon 500 spin columns (30 kDa cutoff, Sartorius), and washed three times with 400 µl UA buffer (8M urea plus 100 mM triethylammonium bicarbonate). Proteins were incubated with 50 mM dithiothreitol in UA buffer for 1 h at 37°C followed by incubation with 50 mM 2-chloroacetamide in UA buffer in the dark for 20 min at 25°C. Following centrifugation, filters were washed with immunoaffinity purification (IAP) buffer (50 mM MOPS/NaOH pH 7.2 plus 10 mM Na2HPO4 plus 50 mM NaCl) and proteins digested overnight at 37°C with 1:50 lysyl endopeptidase (Lys-C, Wako Pure Chemical Corporation) in IAP buffer. Peptides were collected by centrifugation (Lys-C digested peptides) and the remaining larger peptides on the filters were subjected to digestion with 1:100 glycyl endopeptidase (Glu-C, ThermoFisher) at 25°C overnight (Lys-C + Glu-C digested peptides).

Aroankins T.S., Murali S.K., Fenton R.A, & Wu Q. (2021). The Hydrogen-Coupled Oligopeptide Membrane Cotransporter Pept2 is SUMOylated in Kidney Distal Convoluted Tubule Cells. Frontiers in Molecular Biosciences, 8, 790606.

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PEGylation and Transglutamination of Recombinant G-CSF

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The protein rhG-CSF was a kind gift of Sandoz
(Ljubljana, Slovenia). PEG-aldehyde 20 kDa and PEG-NH₂ 20
kDa were purchased from NOF Corporation (Tokyo, Japan). The H₂N-PEG-COOH 20 kDa was purchased from JenKem Technology USA,
Inc. (Plano, TX). Microbial TGase (mTGase), of Streptomyces
mobaraensis origin (ACTIVA M), was provided by Ajinomoto
Co. (Tokyo, Japan). Carbobenzoxy-l-glutaminyl-glycine and
all of the chemicals and solvents were purchased from Merk (Darmstadt,
Germany). Trypsin and Glu-C of sequencing grade were obtained by Thermo
Fisher Scientific (Waltham, MA). Human G-CSF Instant ELISA Kit was
purchased from Life Technologies (Waltham, MA). Mini-PROTEAN TGX precast
gels 4–15% for SDS-PAGE were obtained from Bio-Rad (Milan,
Italy).

Grigoletto A., Marotti V., Tedeschini T., Campara B., Marigo I., Ingangi V, & Pasut G. (2023). Improving the Therapeutic Potential of G-CSF through Compact Circular PEGylation Based on Orthogonal Conjugations. Biomacromolecules, 24(9), 4229-4239.

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Quantifying IgG1 Oxidation and Deamidation

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Oxidized and deamidated species of IgG1 were determined by peptide mapping under reduced conditions using quantitative UHPLC-MS technique. IgG1 samples at 2 mg/mL were treated with 0.25% w/w of RapiGest SF™ (make: Waters, USA; P/N: 186002123) at 25 °C for 1 h followed by reduction with 10 mM dithiothreitol (DTT) (make: Sigma Aldrich, USA; P/N: D0632) for 1 h. Samples were alkylated using 20 mM imidoacetamide (make: Sigma Aldrich, USA; P/N: I6125) for 1 h at 25 °C. Further the samples were digested enzymatically with Trypsin (make: ThermoFisher Scientific, USA; P/N: 90058) for 12 h at 37 °C followed by GluC (make: ThermoFisher Scientific, USA, P/N: 90054) for 10 h at 25 °C. The reaction was arrested by addition of 1% v/v of formic acid (Mass grade) and samples were centrifuged at 12500 RPM for 25 min at 25 °C. The reduced supernatant was further diluted to 0.2 mg/mL using acetonitrile with 0.1% (v/v) trifluroacetic acid of LC/MS grade (make: Merck, P/N: 1.59014) and 10 μL of this sample was analyzed (n = 3) on Acclaim™ VANQUISH™ C18 column, of 2.2 μm particle size and dimensions of 2.1 mm × 250 mm (P/N 074812-V) using Vanquish Flex Binary UHPLC system and further analyzed on Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (all from Thermo Scientific, USA). The data for samples was analyzed by using Xcalibur 4.0 and BioPharma Finder 3.0 software.

Deokar V., Sharma A., Mody R, & Volety S.M. (2020). Comparison of Strategies in Development and Manufacturing of Low Viscosity, Ultra-High Concentration Formulation for IgG1 Antibody. Journal of Pharmaceutical Sciences, 109(12), 3579-3589.

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VEGFR-2 Antibody Production Protocol

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PNGase F was obtained from New England Biolabs (Ipswich, MA). MS Grade proteases, including Trypsin (TPCK treated), Glu-C, and ChymoTrypsin (TLCK treated), Recombinant Protein A Agarose, NuPAGE Novex 4–12% Bis-Tris Protein Gels (1.5 mm, 10-well), and GelCode Blue Staining Reagent were purchased from Thermo Scientific (Waltham, MA). Anti-VEGFR-2/FLK1 antibody against the kinase insert region (cytoplasmic) was generated in-house.^{18 (link)}

Chandler K.B., Leon D.R., Meyer R.D., Rahimi N, & Costello C.E. (2016). Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2. Journal of proteome research, 16(2), 677-688.

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Mass Spectrometry Sample Preparation

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Trifluoroacetic acid (TFA), urea, Triton X-100, α-cyano-4-hydroxycinnamic acid (CHCA) and 2, 5-dihydroxybenzoic acid (DHB) were purchased from Sigma-Aldrich Corp. (St. Louis, MO). CHCA and DHB were recrystallized before use. Bovine lenses were purchased from Pel-Freez Biologicals (Rogers, AR) and stored at −80°C until further use. MS grade trypsin, GluC and Lys C protease were purchased from Thermo Scientific (Rockford, IL). Ammonium formate, formic acid, high-performance liquid chromatography (HPLC)-grade acetonitrile and water were purchased from Fisher Scientific (Pittsburgh, PA).

Wang Z., Ryan D.J, & Schey K.L. (2020). Localization of the Lens Intermediate Filament Switch by Imaging Mass Spectrometry. Experimental eye research, 198, 108134.

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LTBP4 Cleavage by ADAMTS7: Mass Spectrometry

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Purified LTBP4S-A (80 μg) was incubated for 25 h with 70 nm purified ADAMTS7-T8 in 50 mm HEPES, pH 7.5, 5 mm CaCl₂ in the presence or absence of broad-spectrum metalloprotease inhibitor GM6001 (90 μm). Samples were analyzed by SDS-PAGE/Coomassie Blue to confirm proteolysis and the absence thereof in the cleavage and control condition, respectively. New N termini generated by ADAMTS7 were labeled with Tandem Mass Tags (Thermo Fisher) according to the manufacturer's instructions prior to incomplete digest with trypsin (Thermo Fisher), chymotrypsin (Thermo Fisher) and Glu-C (Thermo Fisher). LC-MS/MS was performed at the Proteomic Facility of the University of Liège using an ACQUITY UPLC M-Class System (Waters) hyphenated to a Q Exactive (Thermo Scientific), in nanoelectrospray positive ion mode. Data were analyzed with Proteome Discoverer version. 2.1.1.21. The protein/peptide identifications were performed against a bovine background protein database supplemented with the sequence of the human target protein LTBP4. Search parameters were set as “no Enzyme” because of the specific multi-enzymatic limited digestion that was applied. Scissile bonds were derived from peptides labeled with TMT at the N terminus that were identified in the active protease condition, but not in the control condition. All reported peptides have a false discovery rate equal or lower than 0.01.

Colige A., Monseur C., Crawley J.T., Santamaria S, & de Groot R. (2019). Proteomic discovery of substrates of the cardiovascular protease ADAMTS7. The Journal of Biological Chemistry, 294(20), 8037-8045.

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Protein Reduction and Alkylation for Mass Spectrometry

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Air-dried protein pellets were solubilized in freshly prepared 9 M urea in 50 mM Tris pH 8.0. Ten millimolar aqueous TCEP was added to reduce the disulfide bonds for 30 min in the dark at room temperature. Five millimolar aqueous iodoacetamide was added to alkylate the sulfhydryl groups for 15 min in the dark at room temperature. The solutions were diluted with 50 mM Tris pH 8.0 to 2 M urea and digested with sequencing grade porcine trypsin (Thermo) or Glu-C (Thermo) at a ratio of 20:1 protein-to-protease for 18 to 24 h at 37 °C, shaken at 600 rpm. After digestion, the samples were acidified with 5% formic acid and debris eliminated at 21,100×g for 30 min.

Nguyen K.K., Thurmond S., Hai R, & Genereux J.C. (2019). Protein profiling and pseudo-parallel reaction monitoring to monitor a fusion-associated conformational change in hemagglutinin. Analytical and bioanalytical chemistry, 411(19).

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