For this, 20 µL of the spent medium were analyzed in a HPLC-DAD (Waters 600E, Waters 996) as described by Belchí-Navarro et al. (2012). In addition, 50 mg of freeze-dried cells were extracted overnight in 4 mL methanol at 4°C. The cell extract was diluted with water to a final concentration of 80% (v/v) methanol. Then, 20 µl of the diluted extracts was filtered (Anopore 0.2 µm) and analyzed in a HPLC-DAD (Waters 600E, Waters 996) as described by Bru et al. (2006) using a Spherisorb ODS2 C-18 column (250×4.6 mm, 5 µm). trans-R was identified at 304 nm and quantified by comparison with authentic standard of >99% purity (Sigma-Aldrich, Spain).
Waters 600e
Manufactured by Waters Corporation
Sourced in United States
The Waters 600E is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. The core function of the Waters 600E is to provide precise and reliable solvent delivery for separation, purification, and analysis of a wide range of chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
2414 differential refractometer, by Waters Corporation (1 mentions)
In line degasser, by Waters Corporation (1 mentions)
Waters 717 plus autosampler, by Waters Corporation (1 mentions)
Lc 10at pump, by Shimadzu (1 mentions)
486 tunable absorbance detector, by Waters Corporation (1 mentions)
G4000pwxl, by Tosoh (1 mentions)
Polyethylene glycol, by Merck Group (1 mentions)
C18 column, by Waters Corporation (1 mentions)
Agilent 1260, by Agilent Technologies (1 mentions)
Millennium software, by Waters Corporation (1 mentions)
Dbco peg4 nhs ester, by Vector Laboratories (1 mentions)
Millenium 32, by Waters Corporation (1 mentions)
Dbco amine, by Vector Laboratories (1 mentions)
1450 microbeta trilux liquid scintillation and luminescence counter, by PerkinElmer (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Zq 4000, by Waters Corporation (1 mentions)
Luna c5, by Phenomenex (1 mentions)
V 560, by Jasco (1 mentions)
Lcq deca xp max, by Thermo Fisher Scientific (1 mentions)
Ultra plus microscope, by Zeiss (1 mentions)
19 protocols using waters 600e
1
HPLC-DAD Analysis of Metabolites
2
Quantification of trans-R in Spent Medium and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For this, 20 µL of the spent medium were analyzed in a HPLC-DAD (Waters 600E, Waters 996) as described by Belchí-Navarro et al. (2012) . In addition, 50 mg of freeze-dried cells were extracted overnight in 4 mL methanol at 4°C. The cell extract was diluted with water to a final concentration of 80% (v/v) methanol. Then, 20 µL of the diluted extract was filtered (Anopore 0.2 µm) and analyzed in a HPLC-DAD (Waters 600E, Waters 996) as described by Bru et al. (2006) using a Spherisorb ODS2 C-18 column (250 x 4.6 mm, 5 µm). trans-R was identified at 306 nm and quantified by comparison with authentic standard of >99% purity (Sigma-Aldrich, Spain).
3
Molecular Weight Distribution Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The molecular weight distribution was determined using size-exclusion chromatography (SEC). The system consisted of a controller (Waters 600E, Waters, Milford, MA, USA) connected to a refractive index detector (Waters 2414 Differential Refractometer) and UV detector (Waters 486 Tunable Absorbance Detector) set to 234 nm. The column used was a TSKgel (G4000PWXL, TOSOH Bioscience GmbH, Griescheim, Germany) and the eluent was deionized water, which was pumped using a Waters 600 gradient pump at a flow rate of 0.5 mL/min and degassed using a Waters in-Line degasser. The injection volume was 20 µL, which was performed using an autosampler (Waters 717 plus autosampler). The standards used were polyethylene glycol (400 Da, Merck Schuchardt OHG, Germany) and dextran (2000, 500, 100, 150, 60, 10 and 4 kDa Merck Schuchardt OHG, Germany). The same standards, instrument parameters and column were used for an alkali SEC (100 mM NaOH eluent) with a Shimadzu (Shimadzu Corp., Kyoto, Japan) system (SIL-10AXL autosampler, LC-10AT pump, CTO-10A column oven, RID-10A refractive index detector and SPD-10AV UV-detector).
4
Enzymatic Metabolism of Host Volatiles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The capability of recombinant SzeaGSTd1 to metabolize host volatiles (capryl alcohol, vanillin, and benzaldehyde) was analyzed using high-performance liquid chromatography (HPLC) on the Waters 600E equipment (Waters Corporation, Milford, MA, USA). For this purpose, the total 500 μL volume reaction system containing 100 mM of PBS (pH 7.2), 2.5 mM of GSH, 20 μg of recombinant SzeaGSTd1, and 1 μg/mL of vanillin or 1 mM capryl alcohol or benzaldehyde was incubated at 30 °C with shaking at 200 rpm for 0.5 h; then, 500 μL methanol (HPLC grade) was added to stop the reaction. Subsequently, the reaction mixture was centrifuged at 12,000× g for 20 min, and 500 μL of supernatant was filtered into HPLC vials via a 0.22 mm organic membrane. When the sample (20 μL) was injected into a C18 column (4.6 × 250 mm, 5 µm, Waters Corporation, Milford, CT, USA), the residual content of host volatiles was analyzed with the mobile phase of 80% acetonitrile and 20% water with a 1 mL/min flow rate at 30 °C. The absorbance wavelength for capryl alcohol was set to 206 nm, that for vanillin was set to 230 nm, and that for benzaldehyde set to 237 nm. A heat-inactivated enzyme was used as the control. The experiments were performed with three replicates.
5
Purification of Fraction S5 by HPLC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fraction S5 was dissolved in methanol and filtered, then further separated and purified by semi-preparative HPLC (Waters 600E, Milford, America). Chromatographic conditions were as follows: semi-preparative AQ-C18 Column (10 × 250 mm, 10 μm, Ultimate); the mobile phase of acetonitrile: water = 15:85 (v: v); flow rate of 1.5 mL/min; temperature of 25 °C; detection wavelength of 213 nm; 100 μL per injection. The eluate was collected corresponding to the absorption peak and the purity was assessed using analytical HPLC (Agilent 1260, Palo Alto, America).
6
Reversed Phase HPLC Analysis of Casein and Whey
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Casein fractions and whey proteins were determined by the Reversed Phase -High Performance Liquid Chromatography (RP-HPLC) method [21 (link)] on a HPLC system consisting of a pump capable of mixing four solvents (Waters 600E, Waters, Milford, MA, USA), a photodiode array detector (Waters 996), a helium degasser, a Rheodyne 7125 injector (Rheodyne Inc., Cotati, CA, USA) and Millennium software (v.3.05.01, Waters, Milford, MA, USA).
7
Radiolabeling of Cetuximab for PET Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless specified, all chemicals were purchased
from Sigma-Aldrich
Chemical Co. (St. Louis, MO). Copper-64 was purchased from Washington
University (St. Louis, MO) and the University of Wisconsin (Madison,
WI). DBCO-PEG4-NHS ester and DBCO-amine were purchased
from Click Chemistry Tools (Scottsdale, AZ). CB-TE1K1P-PEG4-DBCO, CB-TE1A1P-DBCO, and 3-azidopropionic acid succinimidyl ester
were synthesized as previously reported.22 Centricon 100 concentrators were purchased from Amicon (Beverly,
MA). Zeba spin desalting columns were from Thermo Scientific (Rockford,
IL). Cetuximab was obtained from ImClone Systems Incorporated (New
York, NY). Size-exclusion HPLC was performed on a Superose 12 HR 10/300
column (Amersham Biosciences, Uppsala, Sweden), attached to a Waters
600E (Milford, MA) chromatography system with a Waters 991 photodiode
array detector and an Ortec model 661 (EG&G Instruments, Oak Ridge,
TN) radioactivity detector. The mobile phase was 20 mM HEPES, 150
mM NaCl, pH 7.3 eluted at a flow rate of 0.5 mL/min. Millenium 32
software (Waters, Milford, MA) was used in analyzing the HPLC chromatograms.
MultiScreen 96-well microtiter plates for receptor binding assays
were counted on a 1450 Microbeta Trilux Liquid Scintillation and Luminescence
counter (PerkinElmer Life Sciences).
from Sigma-Aldrich
Chemical Co. (St. Louis, MO). Copper-64 was purchased from Washington
University (St. Louis, MO) and the University of Wisconsin (Madison,
WI). DBCO-PEG4-NHS ester and DBCO-amine were purchased
from Click Chemistry Tools (Scottsdale, AZ). CB-TE1K1P-PEG4-DBCO, CB-TE1A1P-DBCO, and 3-azidopropionic acid succinimidyl ester
were synthesized as previously reported.22 Centricon 100 concentrators were purchased from Amicon (Beverly,
MA). Zeba spin desalting columns were from Thermo Scientific (Rockford,
IL). Cetuximab was obtained from ImClone Systems Incorporated (New
York, NY). Size-exclusion HPLC was performed on a Superose 12 HR 10/300
column (Amersham Biosciences, Uppsala, Sweden), attached to a Waters
600E (Milford, MA) chromatography system with a Waters 991 photodiode
array detector and an Ortec model 661 (EG&G Instruments, Oak Ridge,
TN) radioactivity detector. The mobile phase was 20 mM HEPES, 150
mM NaCl, pH 7.3 eluted at a flow rate of 0.5 mL/min. Millenium 32
software (Waters, Milford, MA) was used in analyzing the HPLC chromatograms.
MultiScreen 96-well microtiter plates for receptor binding assays
were counted on a 1450 Microbeta Trilux Liquid Scintillation and Luminescence
counter (PerkinElmer Life Sciences).
8
Isolation and Purification of C29-Peridinin
9
Synthesis and Purification of atTic20 Peptide
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A peptide corresponding to the 21-amino acid residues located at the N-terminal of the mature atTic20 (N-ASKDVPSSFRFPPMTKKPQWW-C) was synthesized by standard solid-phase Fmoc chemistry procedures on Wang resin, as previously described [33 (link)]. Briefly, HCTU, HOBt, and DIPEA in DMF were used to activate the C-terminus of amino acids, and a 20% piperidine solution in DMF was utilized for Fmoc deprotection. The peptide was purified by a Waters 600E reversed-phase high-performance liquid chromatography (RP-HPLC) system. Peptide identification and purity were examined using analytical RP-HPLC and electrospray mass spectrometry (Waters ZQ4000). A Luna C5 (Phenomenex) column was used for both analytical and semi-preparative RP-HPLC experiments. Concentration of the peptide was determined using Trp absorption at 280 nm.
10
Cathepsin B-Mediated MMAE Release
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Release of MMAE from prodrugs by a recombinant cathepsin B was analyzed using a modified method from previously published work33 (link). Prodrug stock solutions (80 µL, 10 mM) were added to the 1.92 mL cathepsin B (MilliporeSigma, Cat# C8571, Burlington, MA, USA) containing buffer (25 mM acetate, 1 mM EDTA, pH 5, pre-warmed at 37 °C) at the final concentration of 30 nM (cathepsin B)and 40 µM (prodrug). Aliquots (200 µL) were periodically removed and enzymatic activity was stopped by the addition of thioprotease inhibitor E-64 (30 nM in the final solution, MilliporeSigma, Cat# E3132). The samples were centrifuged and the supernatants were analyzed by HPLC (Waters 600 E coupled with Varian prostar detector, Milford, MA, USA). Samples were prepared at 0, 10, 20, 30, 60, and 120 min. Experiments were performed in triplicate.
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!