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Hiapp

Manufactured by Bachem
Sourced in Switzerland

HIAPP is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It provides consistent and reliable separation, detection, and data analysis capabilities for a wide range of chemical and biological samples.

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4 protocols using hiapp

1

Preparation of hIAPP Peptide Stocks

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The hIAPP stocks were prepared as previously described [39 (link)]. To this end, 1 mg of hIAPP (BACHEM AG, Bubendorf, Switzerland) was dissolved in 512 µL 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Thermo Fisher, Kandel, Germany) and stored for 1 h at room temperature with occasional vortexing. After centrifugation at 15,000 rpm in a microcentrifuge (VWR International, Darmstadt, Germany), the upper ~80% of the solution was divided into 30-microliter aliquots. HFIP was evaporated overnight in a fume hood to obtain the dried peptide films. After HFIP evaporation, the dried aliquots were stored at −21 °C until further use. Since the current experiments required comparatively small amounts of hIAPP, the dry peptide films were redissolved in 75 µL HFIP each, vortexed for 30 s, centrifuged for 15 s using an Eppendorf MiniSpin centrifuge (Eppendorf, Hamburg, Germany), and divided into 10-microliter aliquots. These 10-microliter aliquots were dried again as described above and stored at −21 °C. Immediately before each experiment, a dried hIAPP aliquot was slowly brought to room temperature and subsequently dissolved in 10 µL dimethyl sulfoxide (DMSO, Sigma-Aldrich, Steinheim, Germany), vortexed for 30 s, centrifuged for 15 s, and allowed to reach equilibrium for 10 min.
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2

Denaturation and Purification of hIAPP Peptide

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For denaturation and purification of the peptide, 1 mg of hIAPP (BACHEM AG, Bubendorf, Switzerland) was dissolved in 512 µL 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Thermo Fisher GmbH, Kandel, Germany) and stored for 1 h at room temperature with occasional vortexing. Then, the peptide solution was centrifuged (15,000 rpm) at 4 °C for 30 min using a microcentrifuge (VWR International GmbH, Darmstadt, Germany). In the next preparation step, the upper ~80% of the solution was divided into 20 µL aliquots and left in the fume hood overnight in order to evaporate the HFIP and obtain a dried peptide film. The dried aliquots were stored at −20 °C. For concentration adjustment, the dry peptide films were redissolved in HFIP, vortexed for 30 s, centrifuged for 15 s, using an Eppendorf MiniSpin centrifuge (Eppendorf AG, Hamburg, Germany) and subjected to the same treatment as described above.
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3

Beta-TC-6 Cells Culture and hIAPP Treatment

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Beta-TC-6 cells (ATCC, Manassas, VA) were maintained in monolayer cultures in HEPES-buffered Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY) supplemented with 15% heat-inactivated fetal bovine serum (Life Technologies), 100 IU/ml penicillin and 100 μg/ml streptomycin (Life Technologies). Cells were maintained at 37°C humidified atmosphere with 5% CO2. h-IAPP (Bachem, King of Prussia, PA) was dissolved in water and immediately added it to the culture medium of β-cells at various concentrations.
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4

Calcein Efflux from Lipid Vesicles

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Membrane permeability was measured in standard 96-well transparent microtiter plates in a plate reader (Spectrafluor, Tecan, Salzburg, Austria). Peptides (5 μL of 0.2 mM in DMSO) were added to calcein-containing LUVs (195 μL of lipid vesicles (50 μM) in 10 mM Tris–HCl, 100 mM NaCl buffer (pH 7.4)). As positive control human islet amyloid polypeptide (hIAPP; Bachem) 5 μL of a 0.2 mM in DMSO) was added to calcein-containing LUVs. As a negative control, murine IAPP ((mIAPP; Bachem) 5 μL of 0.2 mM in DMSO) was added to calcein-containing LUVs. For blank only, 5 μL DMSO was added to calcein-containing LUVs. Directly after the addition of all components, the microtiter plate was shaken for 10 s. Fluorescence was measured from the top, every 5 min, using a 485 nm excitation filter and a 535 nm emission filter at 25 °C. The maximum leakage at the end of each measurement was determined by adding 1 μL of 10% Triton X-100 to a final concentration of 0.05% (v/v). The release of fluorescent dye was calculated according to Equation (1):
L(t) is the fraction of dye released (normalized to membrane leakage), Ft is the measured fluorescence intensity, and F0 and F100 are the fluorescence intensities at times t = 0 and after addition of Triton X-100, respectively. All membrane leakage assays were performed two times, each in duplicate, on different days, using different IAPP stock solutions.
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