To detect the Slit2 protein expression in the striatum tissue of SHR with different treatments, immunoblot was conducted as described in our previous study [31 (link)]. Total proteins were extracted from the striatal tissues in PRO-PREP™ buffer (iNtRON Bio-technology, Inc., Seongnam, Republic of Korea), and the concentrations of protein were measured according to a modified Bradford’s assay using a spectrophotometer (Hitachi U3000, Tokyo, Japan) at 595 nm. The proteins were separated into a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) via electrophoresis and then transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). After blocking the membrane with 5% nonfat dry milk, antibodies against rat Slit2 (Cat. #:ab7665, Abcam, Waltham, MA, USA), or β-actin (Cat. #: MAB1501, Merck Millipore, Burlington, MA, USA), they were incubated for 2 h with mild shaking. Subsequent incubation of horseradish peroxidase (HRP) conjugated secondary antibody for another hour was performed. For detecting the antigen–antibody complexes, Immobilon Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA) and an imaging analyzer (GE ImageQuant TL 8.1, GE Healthcare Life Sciences, Pittsburgh, PA, USA) were used.
U 3000
Manufactured by Hitachi
Sourced in Japan
The U-3000 is a high-performance spectrophotometer designed for laboratory use. It measures the absorbance or transmittance of light through a sample across a wide range of wavelengths, enabling quantitative analysis of various materials and solutions.
Automatically generated - may contain errors
Lab products found in correlation
Amx 500, by JEOL (5 mentions)
Lcq xp, by Thermo Fisher Scientific (5 mentions)
Av 400, by Bruker (4 mentions)
Kieselgel 60, by Merck Group (4 mentions)
Jnm eca600, by Bruker (3 mentions)
Avance 3 800, by Bruker (3 mentions)
Ft ir fts 135, by Bio-Rad (2 mentions)
Jnm eca600, by JEOL (2 mentions)
Em 400 detecting unit, by Ametek (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ge imagequant tl 8, by GE Healthcare (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Pro prep buffer, by iNtRON Biotechnology (1 mentions)
60 f254 precoated plates, by Merck Group (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Uv vis spectrometer, by Hitachi (1 mentions)
Evans blue dye, by Merck Group (1 mentions)
Fts 135, by Bio-Rad (1 mentions)
N 1000sw, by Eyela (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
36 protocols using u 3000
1
Immunoblot Analysis of Slit2 Protein
2
Comprehensive Analytical Characterization of Compounds
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Proton (1H) and carbon (13C)
NMR spectra were obtained on a Bruker AV 400 (400/100
MHz), Bruker AMX 500 (500/125 MHz), JEOL JNM-ECA600 (600/150 MHz),
or Bruker AVANCE III 800 (800/200 MHz) spectrometer. Chemical shifts
are reported as parts per million (δ) relative to the solvent
peak. Coupling constants (J) are reported in hertz.
Mass spectra were recorded on a Thermo LCQ XP instrument. Optical
rotations were determined on Jasco III in appropriate solvent. UV
spectra were recorded on U-3000 made by Hitachi in methanol or water.
Infrared spectra were recorded on FT-IR (FTS-135) made by Bio-Rad.
Melting points were determined on a Buchan B-540 instrument and are
uncorrected. The crude compounds were purified by column chromatography
on a silica gel (Kieselgel 60, 70–230 mesh, Merck). Elemental
analyses (C, H, and N) were used to determine the purity of all synthesized
compounds, and the results were within ±0.4% of the calculated
values, confirming ≥95% purity.
NMR spectra were obtained on a Bruker AV 400 (400/100
MHz), Bruker AMX 500 (500/125 MHz), JEOL JNM-ECA600 (600/150 MHz),
or Bruker AVANCE III 800 (800/200 MHz) spectrometer. Chemical shifts
are reported as parts per million (δ) relative to the solvent
peak. Coupling constants (J) are reported in hertz.
Mass spectra were recorded on a Thermo LCQ XP instrument. Optical
rotations were determined on Jasco III in appropriate solvent. UV
spectra were recorded on U-3000 made by Hitachi in methanol or water.
Infrared spectra were recorded on FT-IR (FTS-135) made by Bio-Rad.
Melting points were determined on a Buchan B-540 instrument and are
uncorrected. The crude compounds were purified by column chromatography
on a silica gel (Kieselgel 60, 70–230 mesh, Merck). Elemental
analyses (C, H, and N) were used to determine the purity of all synthesized
compounds, and the results were within ±0.4% of the calculated
values, confirming ≥95% purity.
3
Determining Protein Content in Nanoparticles
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The crosslinked or co-encapsulated HSA content of the nanocomposites was determined by the µBCA method after centrifuging 0.1 mL nanoparticle dispersion and removing the supernatant, while the pellet was dissolved in 0.5 mL DMSO. This solution was diluted 10-fold with purified water, and incubated for 1 h at 60 °C with the same volume of freshly prepared µBCA reagents mixture. DMSO was added to the calibrating HSA solution with the same ratio. After cooling the colored mixtures to room temperature for 20 min, their absorbance was evaluated by spectrophotometry at 562 nm (Hitachi U-3000, Tokyo, Japan).
4
Quantifying Photosynthetic Pigments
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Concentrations of carotenoid and chlorophyll were measured using the method of Lichtenthaler and Buschmann [29 (link)], with the necessary modifications. After combining an aliquot (0.10 g) of the powdered material with 5 mL of 95% ethanol, the mixture was left to cure for 24 h in the dark. Using a spectrophotometer (Hitachi U-3000; Hitachi, Ltd., Chiyoda, Tokyo, Japan), chlorophyll extracts were filtered and examined. The following are the UV absorption wavelengths of total carotenoids, Chl a, and Chl b. The absorption peak of chlorophyll b is measured at 649 nm, total carotenoids at 470 nm, and chlorophyll an at 665 nm. The amounts of total carotenoids, also known as carotenoids, and the concentrations of Chl a (mg/g), Chl b (mg/g), and Chl a/b (mg/g) are also recorded.
5
Analytical Characterization of Synthesized Compounds
6
Absorption Spectroscopy of Ruthenium Complexes
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UV-vis absorption spectrometer (Hitachi U-3000): 280–300 nm; [π → π*] transition of alkynyl (phenyl groups attached to the DDSQ core) and Tpy ligand. 400–600 nm; the metal-to-ligand charge-transfer (MLCT) for [Ru(Tpy)2]2+.
7
Anthocyanin Content Quantification
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To determine anthocyanin content, we employed a modified method based on a previously established protocol [41 (link)]. The absorbance of the supernatant was measured at 530 nm and 657 nm using a spectrophotometer (U-3000, HITACHI, Hitachi City, Japan). The relative anthocyanin content was calculated using the formula: 1 unit = (A530 − 0.25 × A657)/Fresh Weight (g).
8
Turbidity Measurement by Spectrophotometry
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Turbidity (λ = 600 nm, 37 °C) was measured by using an ultraviolet visible light spectrophotometer (HITACHI, Tokyo, Japan, U-3000). The sample cuvette was fluent agitated. An elevation in turbidity meant precipitation of solids.
9
Characterization of Conjugated Nanoparticles
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UV–vis spectra (350 – 600 nm, 1 nm resolution) of the conjugated nanoparticles dispersed in PBS
(1 mg/ml) were recorded in 1 cm quartz
cells with a U-3000 Hitachi, UV–vis spectrometer. For transmission electron
microscopy (TEM) characterization a 4 µl droplet of nanoparticle
suspension was placed on a plain carbon-coated copper TEM grid and allowed to
evaporate in air under ambient laboratory conditions for several hours. Bright
field TEM images were obtained using a TEM (Philips CM12, FEI Ltd, UK) operating
at 80 kV fitted with an X-ray microanalysis detector (EM-400
Detecting Unit, EDAX UK) utilizing EDAX's Genesis software. Typical magnification
of the images was × 100,000. Images were
recorded using a SIS MegaView III digital camera (SIS Analytics, Germany) and
analyzed with the software ImageJ.
(1 mg/ml) were recorded in 1 cm quartz
cells with a U-3000 Hitachi, UV–vis spectrometer. For transmission electron
microscopy (TEM) characterization a 4 µl droplet of nanoparticle
suspension was placed on a plain carbon-coated copper TEM grid and allowed to
evaporate in air under ambient laboratory conditions for several hours. Bright
field TEM images were obtained using a TEM (Philips CM12, FEI Ltd, UK) operating
at 80 kV fitted with an X-ray microanalysis detector (EM-400
Detecting Unit, EDAX UK) utilizing EDAX's Genesis software. Typical magnification
of the images was × 100,000. Images were
recorded using a SIS MegaView III digital camera (SIS Analytics, Germany) and
analyzed with the software ImageJ.
10
Oxidative Stress Biomarkers Evaluation
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The myeloperoxidase (MPO) activity was measured spectrophotometrically with o-dianisidine (spectrophotometer Hitachi U-3000) [27 (link)]. The ferric reducing ability of plasma (FRAP) was measured by spectrophotometry (595 nm) using tripyridyltriazine (TPTZ) and ferric chloride (FeCl3) [28 (link)].
Protein thiol groups were measured spectrophotometrically using DTNB (5,5′-dithiobis 2-nitrobenzoic acid) as described elsewhere [17 (link)]. The molar extinction coefficient (ε) of 1.36 × 104 M−1·cm−1 was used to calculate the thiol contents. Protein carbonyl groups were measured by spectrophotometry using 2,4-dinitrophenylhydrazine as previously described [29 (link)]. Calculations were done using the molar extinction coefficient (ε) of 2.20 × 104 M−1·cm−1.
Albumin, total protein, creatinine, and activities of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured in the plasma to evaluate liver and kidney damage using commercial kits (Gold Analisa®).
Protein thiol groups were measured spectrophotometrically using DTNB (5,5′-dithiobis 2-nitrobenzoic acid) as described elsewhere [17 (link)]. The molar extinction coefficient (ε) of 1.36 × 104 M−1·cm−1 was used to calculate the thiol contents. Protein carbonyl groups were measured by spectrophotometry using 2,4-dinitrophenylhydrazine as previously described [29 (link)]. Calculations were done using the molar extinction coefficient (ε) of 2.20 × 104 M−1·cm−1.
Albumin, total protein, creatinine, and activities of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured in the plasma to evaluate liver and kidney damage using commercial kits (Gold Analisa®).
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