Fig 1 - uploaded by Hans J. Schnittler
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Spectra of Amido Black 10B (12 µg/ml) in the acidic dissolution solution I diss (dotted line) and in the alkaline elution solution II elut (continuous line). Note the isosbestic point at 620 nm and the absorbance maximum of the acidic sample at 672 nm& / f i g. c :
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The determination of total protein is often a key step for the quantitative analysis of various parameters in tissue and general biochemical research. The classical protocols are restricted to a few compatible buffers, and protocols for the determination of protein in solutions containing protein agglomerates or of protein immobilized on solid surf...
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... of the Amido Black absorption, spectra were run in the acidic dissolu- tion solution (I diss ) and compared with spectra recorded in the basic elution solution (II elut ), each against the cor- responding solutions alone. The concentrations of Ami- do Black tested was between 3,25 µg/ml and 15 µg/ml. An isosbestic point is present at 620 nm ( Fig. 1), render- ing absorbance values recorded at this wavelength di- rectly and quantitatively comparable, and excluding any pH-effect. Maximal absorbance in the acidic solution I diss is, however, found at 672 nm, exceeding the value found at 620 nm by 27%. Nevertheless, all spectrophoto- metric recordings documented here were taken at 620 ...
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... The membrane was washed and imaged with chemiluminescence. Band density representing the protein of interest's molecular weight were quantified via colorimetric analysis using ImageJ (NIH, Bethesda, MD) and standardized to total protein concentrations determined using Amido Black (Bio-Rad, Hercules, CA) protein stain 75 . ...
Obesity is a complex disease associated with augmented risk of metabolic disorder development and cellular dysfunction in various species. The goal of the present study was to investigate the impacts of obesity on the metabolic health of old mares as well as test the ability of diet supplementation with either a complex blend of nutrients designed to improve equine metabolism and gastrointestinal health or L-carnitine alone to mitigate negative effects of obesity. Mares (n = 19, 17.9 ± 3.7 years) were placed into one of three group: normal-weight (NW, n = 6), obese (OB, n = 7) or obese fed a complex diet supplement for 12 weeks (OBD, n = 6). After 12 weeks and completion of sample collections, OB mares received L-carnitine alone for an additional 6 weeks. Obesity in mares was significantly associated with insulin dysregulation, reduced muscle mitochondrial function, and decreased skeletal muscle oxidative capacity with greater ROS production when compared to NW. Obese mares fed the complex diet supplement had better insulin sensivity, greater cell lipid metabolism, and higher muscle oxidative capacity with reduced ROS production than OB. L-carnitine supplementation alone did not significantly alter insulin signaling, but improved lipid metabolism and muscle oxidative capacity with reduced ROS. In conclusion, obesity is associated with insulin dysregulation and altered skeletal muscle metabolism in older mares. However, dietary interventions are an effective strategy to improve metabolic status and skeletal muscle mitochondrial function in older mares.
... The membrane was washed and imaged with chemiluminescence. Band density representing the protein of interest's molecular weight were quanti ed via colorimetric analysis using ImageJ (NIH, Bethesda, MD) and standardized to total protein concentrations determined using Amido Black (Bio-Rad, Hercules, CA) protein stain.92 Statistical Analysis Statistical analyses were conducted using GraphPad Prism 9.3.1 software. ...
Obesity is a complex disease associated with augmented risk of metabolic disorder development and cellular dysfunction in various species. The goal of the present study was to investigate the impacts of obesity on the metabolic health of old mares as well as test the ability of diet supplementation with either a complex blend of nutrients designed to improve equine metabolism and gastrointestinal health or L-carnitine to mitigate negative effects of obesity. Mares (n = 19, 17.9 ± 3.7 years) were placed into one of three group: normal-weight (NW, n = 6), obese (OB, n = 7) or obese fed a complex diet supplement for 12 weeks (OBD, n = 6). After 12 weeks, OB mares received L-carnitine alone for 6 weeks. Obesity in mares was significantly associated with insulin dysregulation, reduced (p < 0.05) muscle mitochondrial function, and decreased (p < 0.05) skeletal muscle oxidative capacity with greater (p < 0.05) ROS production when compared to NW. Obese mares fed the complex diet supplement had better insulin sensivity (p < 0.05), greater (p < 0.05) cell lipid metabolism, and higher (p < 0.05) muscle oxidative capacity with reduced (p < 0.05) ROS production than OB. L-carnitine supplementation alone did not significantly alter insulin signaling, but improved (p < 0.05) lipid metabolism and muscle oxidative capacity with reduced (p < 0.05) ROS. In conclusion, obesity is associated with insulin dysregulation and altered skeletal muscle metabolism in older mares. However, dietary interventions are an effective strategy to improve metabolic status and skeletal muscle mitochondrial function in older mares.
... After extraction, tissues were homogenized with a glass homogenizer in radioimmunoprecipitation assay buffer (RIPA). Protein amounts in each sample were determined by the BCA method [15]. Expression levels of HCN1 (MBP, #78896, Cell signaling, US), HCN2 (#30835, Cell signaling, US), HCN4 (#9145, Cell signaling, US) in isolated tissues were determined by western blot. ...
In this study we used ivabradine (IVA), a hyperpolarization-activated cyclic nucleotide–gated (HCN) channel blocker, to identify its effect on spike-wave discharges (SWDs); and aimed to determine the role of IVA on the effects of T-type calcium channel blocker NNC 55-0396, GABAA receptor agonist muscimol and antagonist bicuculline in male WAG/Rij rats. After tripolar electrodes for electrocorticogram (ECoG) recordings were placed on the WAG/Rij rats' skulls, 5, 10, and 20 mg/kg IVA were intraperitoneally administered for 7 consecutive days and ECoG recordings were obtained on days 0th, 3rd, 6th, and 7th for three hours before and after injections. While acute injection of 5, 10, and 20 mg/kg IVA did not affect the total number and the mean duration of SWDs, subacute administration (7 days) of IVA decreased the SWDs parameters 24 hours after the 7th injection. Interestingly, when IVA was administered again 24 hours after the 6th IVA injection, it increased the SWDs parameters. Western-blot analyses showed that HCN1 and HCN2 expressions decreased and HCN4 increased in the 5-month-old WAG/Rij rats compared to the 1-month-old WAG/Rij and 5-month-old native Wistar rats, while subacute IVA administration increased the levels of HCN1 and HCN2 channels, except HCN4. Subacute administration of IVA reduced the antiepileptic activity of NNC, while the proepileptic activity of muscimol and the antiepileptic activity of bicuculline were abolished. It might be suggested that subacute IVA administration reduces absence seizures by changing the HCN channel expressions in WAG/Rij rats, and this affects the T-type calcium channels and GABAA receptors.
... The samples were solubilized by homogenization by up/down pipetting in a 100 µL tip and heated at 96 • C for 10 min [25]. The protein concentration of these retina samples was determined as described [67]. Retinal lysates (~50 µg total protein per lane) were separated by 5% acrylamide SDS-PAGE. ...
Ribbon synapses reliably transmit synaptic signals over a broad signalling range. Rod photoreceptor ribbon synapses are capable of transmitting signals generated by the absorption of single photons. The high precision of ribbon synapses emphasizes the need for particularly efficient signalling mechanisms. Synaptic ribbons are presynaptic specializations of ribbon synapses and are anchored to the active zone. Synaptic ribbons bind many synaptic vesicles that are delivered to the active zone for continuous and faithful signalling. In the present study we demonstrate with independent antibodies at the light-and electron microscopic level that rabconnectin-3α (RC3α)-alternative name Dmx-like 2 (DMXL2)-is localized to the synaptic ribbons of rod photoreceptor synap-ses in the mouse retina. In the brain, RC3α-containing complexes are known to interact with important components of synaptic vesicles, including Rab3-activating/inactivating enzymes, priming proteins and the vesicular H +-ATPase that acidifies the synaptic vesicle lumen to promote full neu-rotransmitter loading. The association of RC3α/DMXL2 with rod synaptic ribbons of the mouse retina could enable these structures to deliver only fully signalling-competent synaptic vesicles to the active zone thus contributing to reliable synaptic communication.
... Quantitative western blots using total cell extracts ( Figure 1) were performed under standard conditions; prior to gel loading, total protein was determined in SDS-dissolved samples, 26 and defined amounts of protein were loaded on each lane. Confluent cultures of HUVEC were pooled (125 cm 2 total) and subsequently divided in five different samples, each used for quantitative immunoprecipitation (IP) of VE-cadherin, a-catenin, b-catenin, ZO-1, and PECAM-1 followed by western blotting. ...
... Protein precipitates are collected in a filter and stained with the dye Amido Black B. The dye is then eluted from the filter and quantified by absorption in the visible wavelength region. Due to the partial purification of the proteins in the sample, this method is significantly more robust than previous ones, making it adequate for protein quantification in complex matrices, namely those enriched in lipids such as whole cells or biomembranes [17][18][19][20] . The original method includes the use of methanol, and this is maintained in all modified protocols introduced 15, 18, 21 . ...
The amount of protein in complex matrices such as food products is an important characteristic, both at a nutritional and pedagogical level. There are several methods available for protein quantification, from simple UV absorption to mass spectrometry. The most common are based on the interaction of the proteins with colored compounds followed by colorimetric quantification. This work uses an approach in which the protein is first partially purified by precipitation, followed by interaction with Amido Black and colorimetric quantification. This allows the quantification of protein content in several food matrices of increasing complexity. The method used is visually very appealing, raising the interest of students from early ages. On the other hand, the interpretation of the results obtained in the different steps involved is an interesting challenge for higher level students.
... Protein lysates were solubilized by homogenization by up-/down pipetting in a 100 µl tip in Laemmli buffer and heated at 96 • C for 10 min. The protein concentration of retina samples dissolved in the Laemmli buffer was determined with an amido black-based quantification method, as described (Dieckmann-Schuppert and Schnittler, 1997). Fifty microgram of retinal lysate was loaded per lane and separated by 10% acrylamide SDS PAGE. ...
Synaptic ribbons are presynaptic specializations that define eponymous ribbon synapses. Synaptic ribbons are largely composed of RIBEYE, a protein containing an N-terminal A-domain and a carboxyterminal B-domain that is identical with CtBP2, a NAD(H)-binding transcriptional co-repressor. Previously we showed that synaptic ribbons are completely absent in RIBEYE knockout mice in which the RIBEYE A-domain-encoding exon had been deleted, but CtBP2 is still made, demonstrating that the A-domain is required for synaptic ribbon assembly. In the present study, we asked whether the RIBEYE B-domain also has an essential role in the assembly of synaptic ribbons. For this purpose, we made use of RIBEYE knockin mice in which the RIBEYE B-domain was replaced by a fluorescent protein domain, whereas the RIBEYE A-domain was retained unchanged. We found that replacing the RIBEYE B-domain with a fluorescent protein module destabilizes the resulting hybrid protein and causes a complete loss of synaptic ribbons. Our results thus demonstrate an essential role of the RIBEYE B-domain in enabling RIBEYE assembly into synaptic ribbons, reinforcing the notion that RIBEYE is the central organizer of synaptic ribbons. This article is protected by copyright. All rights reserved.
... Protein lysates were solubilized by homogenization by up-/down pipetting in a 100 µl tip in Laemmli buffer and heated at 96 • C for 10 min. The protein concentration of retina samples dissolved in the Laemmli buffer was determined with an amido black-based quantification method, as described (Dieckmann-Schuppert and Schnittler, 1997). Fifty microgram of retinal lysate was loaded per lane and separated by 10% acrylamide SDS PAGE. ...
... After sonication lysate was kept in ice for 15 min and followed by centrifugation at 10,000× g for 10 min at 4 °C. Supernatant was collected and protein concentration was determined by the Amido Black method as described by [147]. Equal protein amount (30 µ g) of the retinal lysates from CFA control and MOG/CFA immunized mice retina was separated by 10% SDS-PAGE followed by wet transfer on to nitrocellulose membrane in the cold room at 50 V for 5 h. ...
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that finally leads to demyelination. Demyelinating optic neuritis is a frequent symptom in MS. Recent studies also revealed synapse dysfunctions in MS patients and MS mouse models. We previously reported alterations of photoreceptor ribbon synapses in the experimental auto-immune encephalomyelitis (EAE) mouse model of MS. In the present study, we found that the previously observed decreased imunosignals of photoreceptor ribbons in early EAE resulted from a decrease in synaptic ribbon size, whereas the number/density of ribbons in photoreceptor synapses remained unchanged. Smaller photoreceptor ribbons are associated with fewer docked and ribbon-associated vesicles. At a functional level, depolarization-evoked exocytosis as monitored by optical recording was diminished even as early as on day 7 after EAE induction. Moreover compensatory, post-depolarization endocytosis was decreased. Decreased post-depolarization endocytosis in early EAE correlated with diminished synaptic enrichment of dynamin3. In contrast, basal endocytosis in photoreceptor synapses of resting non-depolarized retinal slices was increased in early EAE. Increased basal endocytosis correlated with increased de-phosphorylation of dynamin1. Thus, multiple endocytic pathways in photoreceptor synapse are differentially affected in early EAE and likely contribute to the observed synapse pathology in early EAE.
... The remaining hemisphere was homogenized with the liquid nitrogen and treated with RIPA buffer for western blot analysis. Total protein levels in the PBS-homogenized and RIPA-homogenized samples were determined by Lowry and bicinchoninic acid (BCA) methods, respectively [34,35]. ...
Bipolar disorder (BD) is a multifactorial chronic and refractory disease characterized by manic, depressive, and mixed mood episodes. Although epidemiological, and pathophysiological studies demonstrated a strong correlation between bipolar disorder and oxidative stress, precise etiology is still missing. Recent studies suggested the possible role of transient receptor potential channels (TRP) in the BD but, current knowledge is limited. Therefore, the current study investigates the possible role of TRPV1 in the ouabain-induced model of BD. The model was created with intracerebroventricular single dose ouabain (10⁻³ M) administration. Animals were treated with capsaicin, capsazepine, and lithium for seven days. Mania and depressive-like states were investigated with open-field, sucrose preference, and elevated plus maze tests. Oxidative stress was assessed by measuring total antioxidant and oxidant states, spectrophotometrically. The phosphorylation Glycogen synthase kinase-3β (GSK-3β) evaluated by western blotting. Our results demonstrated that capsaicin dose-dependently inhibited the ouabain-induced hyperlocomotion and depression. Although capsazepine exacerbated behavioral impairment, it did not show a significant effect on the antioxidant and oxidant states, and the effects of capsazepine on behaviors were abolished by combination with capsaicin. Additionally, capsaicin potently prevented the ouabain-induced decrease in GSK-3β phosphorylation. In contrast, capsazepine potentiated ouabain-induced decrease in GSK-3β phosphorylation and combination with capsaicin, suppressed the effect of capsazepine on GSK-3β phosphorylation. The effects of TRPV1 activation on oxidative stress and mania-like behaviors in the ouabain-induced BD model might be regulated by GSK-3β phosphorylation.
