Perifusion experiments were performed by the University of Pennsylvania Diabetes Research Center (P30-DK19525). Briefly, islets were hand-picked and cultured for 3–4 days. Islets were perifused with Krebs bicarbonate buffer (2.2 mM Ca2+, 0.25% bovine serum albumin, 10 mM HEPES [acid], and 95% O2 and 5% CO2 equilibration, pH 7.4) to reach baseline hormone secretion values before the addition of the appropriate secretagogues. Samples were collected at regular intervals with a fraction collector (Waters Corporation) and insulin content was determined using a radioimmunoassay (University of Pennsylvania Diabetes Research Center).
Fraction collector
Manufactured by Waters Corporation
Sourced in United States
The Fraction Collector is a laboratory instrument designed to automatically collect and store fractions of a sample during chromatographic separation processes. It is used to separate and collect specific components of a mixture for further analysis or purification.
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Lab products found in correlation
5 protocols using fraction collector
1
Perifusion Assay for Insulin Secretion
2
Metabolite Extraction and HPLC Analysis
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Blood samples and brains were collected from mice immediately postscan,
and an acetonitrile/PBS (1:1) mixture was added at twice the volume
of each sample. The blood solution was vortexed, and brains were homogenized
with a pellet pestle, followed by centrifugation at 3000g for 10 min. After the supernatant had been separated from the pellet,
each portion was counted with a gamma counter (PerkinElmer Wizard
2480) to determine the extraction efficiency. Part of the supernatant
(>200 μL) was diluted with the same volume of water and then
filtered with a 0.45 μm nylon filter, and 200 μL of the
filtered supernatant was injected into an analytical metabolite HPLC
instrument (1200 series, Agilent Technologies) equipped with an Agilent
SB-C18 column (5 μm, 25 cm × 4.6 mm) and a fraction collector
(Waters). The gradient mobile phase consisted of acetonitrile and
a 0.1% TFA solution, and the flow rate was 1 mL/min. A fraction was
collected every 30 s and counted with the gamma counter or directly
with the LabLogic Laura Posi-RAM radiodetector.
and an acetonitrile/PBS (1:1) mixture was added at twice the volume
of each sample. The blood solution was vortexed, and brains were homogenized
with a pellet pestle, followed by centrifugation at 3000g for 10 min. After the supernatant had been separated from the pellet,
each portion was counted with a gamma counter (PerkinElmer Wizard
2480) to determine the extraction efficiency. Part of the supernatant
(>200 μL) was diluted with the same volume of water and then
filtered with a 0.45 μm nylon filter, and 200 μL of the
filtered supernatant was injected into an analytical metabolite HPLC
instrument (1200 series, Agilent Technologies) equipped with an Agilent
SB-C18 column (5 μm, 25 cm × 4.6 mm) and a fraction collector
(Waters). The gradient mobile phase consisted of acetonitrile and
a 0.1% TFA solution, and the flow rate was 1 mL/min. A fraction was
collected every 30 s and counted with the gamma counter or directly
with the LabLogic Laura Posi-RAM radiodetector.
3
LC-SPE Purification and Prep-HPLC Separation
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The LC-SPE purifications
were performed as previously described50 (link) with modifications of the HPLC method to facilitate
the separation of the current fractions. To accumulate more material,
some of the fractions were re-purified using prep-HPLC coupled to
a PDA detector and a fraction collector (Waters, Milford, MA, USA).
Each of the fraction (70 mg) was diluted with methanol (1 mL). An
X-Bridge preparative C18 column (5 μm, 19 × 250 mm) was
used for the separation. The mobile phase consisted of 0.1% formic
acid in water (solvent A) and methanol (solvent B) and was pumped
at a flow rate of 15 mL/min. Gradient elution was applied as follows:
20% B for 1.7 min, 20–80% B (1.7–15 min), 80% B (15–30
min), 80–95% B (30–33 min), 95% B (33–45 min),
95–100% B (45–52 min), 100% B (52–56 min), 100%–20%
B (56–58 min), and 20% B (58–62 min). The injection
volume was 300 μL. The samples were injected multiple times
to accumulate the material. Data were collected using MassLynx4.1
(Waters, USA) software. The purified compounds were collected using
a fraction collector and subsequently combined and concentrated using
the speed vacuum.
were performed as previously described50 (link) with modifications of the HPLC method to facilitate
the separation of the current fractions. To accumulate more material,
some of the fractions were re-purified using prep-HPLC coupled to
a PDA detector and a fraction collector (Waters, Milford, MA, USA).
Each of the fraction (70 mg) was diluted with methanol (1 mL). An
X-Bridge preparative C18 column (5 μm, 19 × 250 mm) was
used for the separation. The mobile phase consisted of 0.1% formic
acid in water (solvent A) and methanol (solvent B) and was pumped
at a flow rate of 15 mL/min. Gradient elution was applied as follows:
20% B for 1.7 min, 20–80% B (1.7–15 min), 80% B (15–30
min), 80–95% B (30–33 min), 95% B (33–45 min),
95–100% B (45–52 min), 100% B (52–56 min), 100%–20%
B (56–58 min), and 20% B (58–62 min). The injection
volume was 300 μL. The samples were injected multiple times
to accumulate the material. Data were collected using MassLynx4.1
(Waters, USA) software. The purified compounds were collected using
a fraction collector and subsequently combined and concentrated using
the speed vacuum.
4
Semi-Preparative RP-HPLC Purification of KD60
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Semi-preparative RP-HPLC was carried out according to Vilcacundo et al. 19 with some modifications. A Hi-Pore Reversed Phase RP-318 (250 x 21.5 mm) column (Bio-Rad) was used. KD60 was prepared (10 mg/mL), and the injection volume was 400 μL. Fractions were eluted at a flow rate of 10 mL/min, with a linear gradient of solvent B (acetonitrile: trifluoroacetic acid (TFA), 1000:0.8, v/v) in A (water:TFA, 1000:1, v/v) going from 0% to 70% B in 40 min, 70% to 100% B in 15 min, 15 min with 100% B and from 100% B to 0% B in 20 min. Each chromatographic run was repeated 50-55 times, and the fractions automatically collected with a Fraction Collector (Model II, Waters, Mildford, MA, USA) were pooled, lyophilized, and stored at -20°C until further analyses. Their peptide content was determined by the BCA method.
5
Size Exclusion Chromatography Purification of Oligosaccharides
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The freeze-dried material obtained from the depolymerisation reaction was redissolved in 2.5 ml deionised water and fractionated by SECon a BioCAD 700E Workstation FPLC system equipped with a Waters Fraction Collector. Two XK26/100 columns preceded by a XK26/20 guard column (GE healthcare) were packed with Bio-Gel P-10 Fine resin (Bio-Rad Laboratories) and connected in series. Enzymatically cleaved DS tetrasaccharides and hexasaccharides were used as standards. The sample was loaded on a 5 ml loop and was run at a flow rate of 0.4 ml/min for a total of 62.5 h, with PBS as the mobile phase. The UV absorbance was recorded at 218 nm and the fractions containing oligosaccharides were collected, pooled and freeze dried.
Prior to NMR experiments, the lyophilized samples were desalted using the BioCad 700E system equipped with a XK16/40 column packed with Sephadex G25 superfine (Sigma-Aldrich). Each run was carried out with an injection volume of 5 ml and dH 2 O as mobile phase. For each run, the injection loop was first flushed for 6 minutes at 2.5 ml/min and then the flow rate was increased to 4 ml/min for 21 minutes and 3 ml fractions were collected. The UV absorbance was recorded at 218 nm. Four to five fractions were pooled together, yielding desalted oligosaccharides in 12-15 ml of solution. The samples were subsequently freeze-dried.
Prior to NMR experiments, the lyophilized samples were desalted using the BioCad 700E system equipped with a XK16/40 column packed with Sephadex G25 superfine (Sigma-Aldrich). Each run was carried out with an injection volume of 5 ml and dH 2 O as mobile phase. For each run, the injection loop was first flushed for 6 minutes at 2.5 ml/min and then the flow rate was increased to 4 ml/min for 21 minutes and 3 ml fractions were collected. The UV absorbance was recorded at 218 nm. Four to five fractions were pooled together, yielding desalted oligosaccharides in 12-15 ml of solution. The samples were subsequently freeze-dried.
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