SAM, SAH, and the SAM:SAH ratio were determined in brain tissue samples. The brain tissue samples were homogenized using a motor-driven tissue homogenizer (PT1200E, Kinematica, Lucerne, Switzerland). Next, 100 mg extracts of the brain tissue were resuspended in 300 μL 0.4 mol/L perchloric acid. Homogenates were centrifuged at 20,000× g for 10 min at 4 °C. The supernatant was filtered through a 0.45-μm membrane filter (Millipore, Billerica, MA, USA), followed by loading into a Venusil MP-C18 column (4.6 mm × 250 mm, 5 μm particle, Agela Technologies, Wilmington, DE, USA) fitted with a matched guard column, run by HPLC system (Waters, Milford, MA, USA). Absorption of eluted compounds was monitored at λ = 254 nm with an ultraviolet detector. A two-buffer elution system was used: mobile phase A and B, both contained 4 mmol/L 1-heptanesulfonic acid (pH 4) and 10 mmol/L ammonium formate. The mobile phase B contained 50% acentonitrile by volume. Elution of SAM and SAH was achieved at a flow rate of 1 mL/min with the following parameters: 0–0.5 min, 100% A; 0.5–20 min, linear gradient to 75% A and 25% B; 20–30 min, 25% B; 30–45 min, 100% A.
Pt 1200e
Manufactured by Kinematica
Sourced in Switzerland
The PT 1200E is a homogenizer manufactured by Kinematica. It is designed for high-speed dispersing and homogenizing applications in laboratory settings. The device features a powerful motor and robust construction to handle a variety of sample types and volumes.
Automatically generated - may contain errors
Lab products found in correlation
0.45 μm membrane filter, by Merck Group (1 mentions)
Hplc system, by Waters Corporation (1 mentions)
Venusil mp c18 column, by Agela Technologies (1 mentions)
Phosstop, by Roche (1 mentions)
A6885, by Merck Group (1 mentions)
Immulite 2000 xpi, by Siemens (1 mentions)
Sorvall sr70, by Thermo Fisher Scientific (1 mentions)
0.22 μm nitrocellulose filter, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using pt 1200e
1
Brain SAM and SAH Quantification
2
Muscle Homogenate Preparation and cAMP Treatment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole tissue homogenate samples were prepared by homogenizing ~5 mg of frozen EDL muscle (1:50 wt/vol) using a handheld polytron homogenizer (Polytron PT 1200E Kinematica, Lucerne, Switzerland) three times at maximum speed for 5 to 10 s in the ice-cold homogenizing solution (50 mM HDTA, 90 mM HEPES, 126 mM K+, 36 mM Na+, 8 mM ATP, 8.5 mM total Mg2+, 10 mM creatine phosphate (pH 7.10) with phosphatase inhibitor (PhosSTOP; Roche Diagnostics, Mannheim, Germany), 295 ± 10 mosmol/kg H2O). Following homogenization, whole muscle samples were vortexed three times at maximum speed for ~10 s and then diluted to 10 µg/µL in homogenizing solution. The homogenate samples were then divided into three tubes (50 to 100 µL each) for control and cAMP-treatments (cAMP). We performed treatments using sodium-salt cAMP (Sigma A6885, same as fiber Ca2+ experiments). For cAMP treatment, cAMP was added to the whole muscle homogenates at 100 µM, using a stock solution (dissolved in ddH2O). Samples were vortexed for ~10 s then left for 1 min at room temperature (~23 °C). After 1 min, 3XSDS loading buffer was added (2:1 v/v) to all samples, which were then incubated at RT for ~1 h and stored at −80 °C until Western blot analysis.
3
Folate and Homocysteine Levels in Pups
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Angular venous blood was collected from pups in coagulant tubes and then centrifuged at 3000× g for 10 min to obtain serum. The brain tissues of pups were homogenized by a motor-driven tissue homogenizer (PT1200E; Kinematica, Lucerne, Switzerland). Folate concentration was determined using a competitive protein-binding assay and an automated chemiluminescence system (Immulite 2000 Xpi; Siemens, Berlin, Germany) according to the manufacturer’s instructions. The assay detected all types of folates, including folic acid, dihydrofolate, and tetrahydrofolate [20 (link)]. Serum samples were diluted 10 times with 0.9% saline to reach the detection limit of this system, which was 1–24 ng/mL. The total Hcy concentration was quantified using an Auto-Chemistry Analyzer (DIRUI; Changchun, China) and Hcy Reagent (Medical System, Ningbo, China), for which the detection limit was 3–150 μmol/L.
4
Quantification of Aortic Biomarkers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure NO, TAC, BH4, BH2, PDE-3, cGMP, CGRP, and MDA levels, the animals were euthanized, and sections of the thoracic aorta were obtained and immediately frozen in liquid nitrogen and stored at -70°C until used. On the day of analysis, tissue samples were homogenized (Polytron PT 1200 E, Kinematica, NY, USA) in cold 100 mM phosphate buffer at pH 7.4, then centrifuged at 16,000 g during 15 min at 10°C (Sorvall SR70, Thermo Scientific Inc., Urbana, IL, USA). Supernatants were filtered through a 0.22 μm nitrocellulose filter (Millipore, Billerica, MA, USA).
The determination of NO and TAC was performed directly on the filtered supernatant, whereas for BH4, BH2, PDE-3, cGMP, and MDA determinations, supernatants were diluted 1 : 10 with 0.1 M NaOH before being analyzed. All samples were kept at -70°C until the beginning of the respective analysis.
The determination of NO and TAC was performed directly on the filtered supernatant, whereas for BH4, BH2, PDE-3, cGMP, and MDA determinations, supernatants were diluted 1 : 10 with 0.1 M NaOH before being analyzed. All samples were kept at -70°C until the beginning of the respective analysis.
5
Membrane Preparation from S1P Receptor-Expressing Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Membranes were prepared from stable or transient S1P 5 expressing cells after adherent culture in 500 cm 2 culture trays. Cells were detached with cell-lifting buffer (10 mM HEPES, 154 mM NaCl, 6.85 mM EDTA, pH 7.4) and pelleted by centrifugation for 5 minutes at 1000 rpm. Cell pellets were then resuspended and homogenized in membrane preparation buffer (10 mM HEPES and 10 mM EDTA, pH 7.4) using a Polytron PT 1200E homogenizer (Kinematica, Luzern, Switzerland). Cells homogenates were centrifuged at 48,000g at 4 C for 30 minutes to collect the membrane pellet. The supernatant was discarded, and the pellet was rehomogenized and recentrifuged as described above in membrane preparation buffer. The final pellet was collected and homogenized in ice cold resuspension buffer (10 mM HEPES and 0.1 mM EDTA, pH 7.4). Aliquots were stored at À80 C until required for radioligand binding or [ 35 S]-GTPcS binding assays.
6
Microsome Isolation from Liver and Cortex
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!