Islets were isolated from NMRI mice and disrupted by sonication in 10 mM Tris‐HCl buffer. The islet homogenate was centrifuged at 204 000G for 75 minutes at 4°C. The pellet was solubilized in 10 mM Tris‐HCl buffer containing 0.01% Triton X‐100 and 1 μg/mL aprotinin. DPP‐IV activity was determined using the H‐Gly‐Pro‐AMC (Bachem, I‐1225) in the presence and absence of 5 nM sitagliptin. The islet sample was incubated with 10 μM H‐Gly‐Pro‐AMC for 120 minutes at 37°C. The amount of AMC liberated was compared with a 1 μM AMC standard.
H gly pro amc
Manufactured by Bachem
Sourced in Switzerland, Ireland
H-Gly-Pro-AMC is a chemical compound used as a substrate in biochemical assays. It consists of the amino acids glycine and proline, with a fluorescent 7-amino-4-methylcoumarin (AMC) group attached. This compound can be used to measure the activity of enzymes that cleave peptide bonds, such as proteases.
Automatically generated - may contain errors
Lab products found in correlation
Hplc grade acetonitrile acn and water, by Avantor (2 mentions)
Tris glycine sds page buffer, by National Diagnostics (2 mentions)
Protogel stacking buffer, by National Diagnostics (2 mentions)
Protein loading buffer blue, by National Diagnostics (2 mentions)
Quick start bradford 1 dye reagent, by Bio-Rad (2 mentions)
Diprotin a, by Bachem (2 mentions)
Flavourzyme 500 l, by Novozymes (2 mentions)
Alcalase 2.4 l, by Novozymes (2 mentions)
Z gly pro amc, by Bachem (1 mentions)
Enspire, by PerkinElmer (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Corning (1 mentions)
6 protocols using h gly pro amc
1
Islet DPP-IV Activity Assay
2
Recombinant Enzyme Storage and Validation
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Recombinant human enzymes [rhFAP (R&D Systems), rhPREP (R&D Systems) and rhDPPIV (Biolegend)] were stored in stocks at -70°C and diluted as required to be used at final concentration of 0.1 µg/ml. Enzymes were aliquoted upon receipt from the supplier and new stocks made from a fresh aliquot as required. Stocks were made at [4x] and control substrates of Z-Gly-Pro-AMC and H-Gly-Pro-AMC (Bachem) were used to confirm activity for each new stock of enzyme.
3
Quantifying DPP Enzyme Activity
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Cells were washed once with DPBS, incubated in 0.05% Trypsin–EDTA for a few minutes at 37 °C, and harvested. After two washing steps with DPBS, cells were resuspended in 150 μL hypotonic buffer (20 mM HEPES at pH 7.9; 1.5 mM MgCl2; 10 mM KCl; 0.05% Triton-X-100; and 1 mM DTT in ddH2O) and incubated for 10 min on ice before centrifugation at 600 rcf and 4 °C for 6 min. The protein concentration of the supernatant was measured via the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific), according to the protocol. An amount of 5 μg protein was mixed with 95 µL hypotonic buffer and 5 μL of 250 µM fluorogenic DPP peptide substrate H-Gly-Pro-AMC (Bachem, Bubendorf, Switzerland). Enzyme activity was measured every minute for 1 h via the EnSpire (Perkin Elmer, Bridgeville, PA, USA) at 480 nm.
4
DPP4 Inhibition Assay Protocol
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DPP4 activity was monitored through the cleavage of fluorescent substrate H-Gly-Pro-AMC purchased from Bachem (Bubendorf, Switzerland). Active recombinant human DPP4 enzyme derived from an NS0 cell line (residues Asp34-Pro766) was obtained from R&D Systems (Minneapolis, MD). DPP4 inhibitors were purchased from SelleckChem (Houston, TX) and reconstituted in 75% DMSO (Sigma Aldrich, Gillingham, UK) and 25% H 2 O, aliquoted, and stored at -20 °C until use. MSD DPP4 inhibitors were selected from the MSD compound library, 10 mM stocks were prepared in 75% DMSO, 25% H 2 O. Inhibitor titrations were performed in Corning 1536 high-base, white nonbinding plates (Corning, Corning, NY) using 1536 low-base plates for the inhibitor source plates (Brooks, Chelmsford, MA). Time-dependent inhibition assays were run in black nonbinding 384-well plates (Corning) using the same source plates as above (Brooks). Assay buffer comprised 25 mM Tris (Corning) at pH 8, as recommended by R&D Systems, and was used for all enzyme and substrate dilutions.
5
Mussel Byssus Protein Extraction and Analysis
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Mussel (Mytilus edulis) byssus was kindly provided by Fastnet Mussels (Gearhies, Bantry, Co. Cork). ProtoGel®, ProtoGel® Resolving Buffer (4×), ProtoGel® Stacking Buffer (4×), Tris-Glycine-SDS-PAGE Buffer (10×) and Protein Loading Buffer Blue (2×) were obtained from National Diagnostics (Atlanta, USA). Quick Start Bradford 1 × Dye Reagent® was supplied by Bio-Rad (Dublin, Ireland). Bovine lung was kindly provided by Gaelic Meats and Livestock Ltd. (Limerick, Ireland), and H-Gly-Pro-AMC and Diprotin A were obtained from Bachem (Bubendorf, Switzerland). HPLC-grade acetonitrile (ACN) and water were obtained from VWR (Dublin, Ireland). Brewers Clarex® (Aspergillus niger derived prolyl endoproteinase (An-PEP) specific activity: 37 × 10 -3 U/mg) was supplied as a gift from Dutch State Mines (DSM, Heerlen, Netherlands), Corolase® PP was provided by AB Enzymes (Darmstadt, Germany), Alcalase® 2.4 L and Flavourzyme® 500 L were obtained from Novozymes A/S (Bagsvaerd, Denmark), Promod 144MG was kindly provided by Biocatalysts Ltd (Parc Nantgarw, Wales, UK). All other reagents, including collagenase from Clostridium histolyticum (Type 1), were obtained from Sigma Chemical Company Ltd. (Gillingham, UK).
6
Mussel Byssus Protein Extraction and Analysis
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Mussel (Mytilus edulis) byssus was kindly provided by Fastnet Mussels (Gearhies, Bantry, Co. Cork). ProtoGel®, ProtoGel® Resolving Buffer (4×), ProtoGel® Stacking Buffer (4×), Tris-Glycine-SDS-PAGE Buffer (10×) and Protein Loading Buffer Blue (2×) were obtained from National Diagnostics (Atlanta, USA). Quick Start Bradford 1 × Dye Reagent® was supplied by Bio-Rad (Dublin, Ireland). Bovine lung was kindly provided by Gaelic Meats and Livestock Ltd. (Limerick, Ireland), and H-Gly-Pro-AMC and Diprotin A were obtained from Bachem (Bubendorf, Switzerland). HPLC-grade acetonitrile (ACN) and water were obtained from VWR (Dublin, Ireland). Brewers Clarex® (Aspergillus niger derived prolyl endoproteinase (An-PEP) specific activity: 37 × 10 -3 U/mg) was supplied as a gift from Dutch State Mines (DSM, Heerlen, Netherlands), Corolase® PP was provided by AB Enzymes (Darmstadt, Germany), Alcalase® 2.4 L and Flavourzyme® 500 L were obtained from Novozymes A/S (Bagsvaerd, Denmark), Promod 144MG was kindly provided by Biocatalysts Ltd (Parc Nantgarw, Wales, UK). All other reagents, including collagenase from Clostridium histolyticum (Type 1), were obtained from Sigma Chemical Company Ltd. (Gillingham, UK).
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