The Design of a Quantitative Western Blot Experiment

Taylor, Sean C.; Posch, Anton

doi:10.1155/2014/361590

Cited by 296 publications

(206 citation statements)

References 23 publications

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“…For Western blot we extracted the proteins from a pool of 30 embryos or cultured RBCs in radio immunoprecipitation assay buffer (Santa Cruz Biotechnology). To quantify the protein, we used total protein normalization methods using stain-free gels and blot methodology described in detail in SI Materials and Methods (Bio-Rad) (47). Immunogold was performed by standard procedures on 30-nm cryosections.…”

Section: Methodsmentioning

confidence: 99%

Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

Cortí

Xue

Tejero

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The discovery of novel globins in diverse organisms has stimulated intense interest in their evolved function, beyond oxygen binding. Globin X (GbX) is a protein found in fish, amphibians, and reptiles that diverged from a common ancestor of mammalian hemoglobins and myoglobins. Like mammalian neuroglobin, GbX was first designated as a neuronal globin in fish and exhibits six-coordinate heme geometry, suggesting a role in intracellular electron transfer reactions rather than oxygen binding. Here, we report that GbX to our knowledge is the first six-coordinate globin and the first globin protein apart from hemoglobin, found in vertebrate RBCs. GbX is present in fish erythrocytes and exhibits a nitrite reduction rate up to 200-fold faster than human hemoglobin and up to 50-fold higher than neuroglobin or cytoglobin. Deoxygenated GbX reduces nitrite to form nitric oxide (NO) and potently inhibits platelet activation in vitro, to a greater extent than hemoglobin. Fish RBCs also reduce nitrite to NO and inhibit platelet activation to a greater extent than human RBCs, whereas GbX knockdown inhibits this nitrite-dependent NO signaling. The description of a novel, sixcoordinate globin in RBCs with dominant electron transfer and nitrite reduction functionality provides new insights into the evolved signaling properties of ancestral heme-globins.nitrite | nitric oxide | blood | RBC | platelet

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Section: Methodsmentioning

confidence: 99%

Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

Cortí

Xue

Tejero

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…all feedback circuits are positive and the system has a monotone flow [25]) and hence the courses are similar for all three components, the results are shown for ppERK only. The phosphorylation rates k 4 . This reflects the fact that the system shows a quasi-bistable behavior, meaning that the system is monostable but able to maintain a state different from zero for a very long time upon a transient signal and returns to its steady state at zero only at a later time point (for more details on this phenomenon see [15]).…”

Section: Case Study I: Global Sensitivity Analysismentioning

confidence: 99%

“…This second normalization is required for a comparison of data from different replicates. Since Western blotting is also used more and more frequently for a quantitative analysis, several studies are involved with the experimental protocols, testing of linear ranges, and proper normalization procedures [4,5]. In this work we use the term rescaling whenever model parameters and variables are multiplied by constant factors, which is often applied to obtain dimensionless models.…”

Section: Introductionmentioning

confidence: 99%

The effect of model rescaling and normalization on sensitivity analysis on an example of a MAPK pathway model

Kirch

Thomaseth

Jensch

et al. 2016

EPJ Nonlinear Biomed Phys

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Background:The description of intracellular processes based on chemical reaction kinetics has become a standard approach in the last decades, and parameter estimation poses several challenges. Sensitivity analysis is a powerful tool in model development that can aid model calibration in various ways. Results can for example be used to simplify the model by elimination or fixation of parameters that have a negligible influence on relevant model outputs. However, models are usually subject to rescaling and normalization to reference experiments, which changes the variance of the output. Thus, the results of the sensitivity analysis may change depending on the choice of these rescaling factors and reference experiments. Although it might intuitively be clear, this fact has not been addressed in the literature so far. Methods: In this study we investigate the effect of model rescaling and additional normalization to a reference experiment on the outcome of two different sensitivity analyses. Results are exemplified on a model for the MAPK pathway module in PC-12 cell lines. For this purpose we apply local sensitivity analysis and a global variance-based method based on Sobol sensitivity coefficients, and compare the results for differently scaled and normalized model versions. Results: Results indicate that both sensitivity analyses are invariant under simple rescaling of variables and parameters with constant factors, provided that sensitivity coefficients are normalized and that the parameter space is appropriately chosen for Sobol's method. By contrast, normalization to a reference experiment that also depends on parameters has a large impact on the results of any sensitivity analysis, and in particular complicates the interpretation. Conclusion: This work shows that, in order to perform sensitivity analysis, it is necessary to take into account the dependency on parameters of the reference condition when working with normalized model versions.

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“…Intragastric tubes were inserted, and piglets were housed in pairs in cages until euthanasia at Background amplitude-integrated EEG (aEEG) was scored using a modified EEG background and seizure activity scoring system ( Piglets were assessed at 8,12,16,24,48 and 72 h on nine neurologic measures such as level of consciousness, respiration, ability to stand and walk, the righting reflex and presence of clinical seizures as described above (see post-insult monitoring). Each neurologic measure was assigned a score of 2 (normal), 1 (moderately abnormal) or 0 (pathologic).…”

Section: Recovery and Electroencephalographic Monitoringmentioning

confidence: 99%

“…Animals were assessed for neurobehaviour at 8,12,16,24,48 and 72 h as previously described (Thoresen et al 1996, Foster et al 2001. Animals were assessed on nine neurologic measures such as level of consciousness, respiration, ability to stand and walk, the righting reflex and presence of clinical seizures as described above (see Appendix 2 of thesis).…”

Section: Neurobehavioural Scoringmentioning

confidence: 99%

Examining the GABA<sub>A</sub> receptor alpha-subunits in the neonatal pig brain: changes across development and the effect of seizures after hypoxic-ischaemic brain injury

Miller¹

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Examining the GABAA receptor α-subunits in the neonatal pig brain:Changes across development and the effect of seizures after hypoxic-ischaemic brain injury. Stephanie Melita Miller BSc (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017Faculty of Medicine i ABSTRACT Hypoxic ischaemic encephalopathy (HIE) is the most common cause of mortality and morbidity in the newborn. Reduced blood flow to the foetus, and consequently the foetal brain, due to maternal factors (e.g. hypertension) or foetal factors (e.g. umbilical cord compression); leads to significant brain injury and neurodevelopmental disability. HIE remains the leading cause of seizures in the term and preterm infant, accounting for over 60% of all seizures in the newborn period. Current antiepileptic drugs (AEDs) are largely ineffective in the newborn, however there has been little change to treatments for neonatal seizures over the last 50 years. AEDs achieve good seizure control in children and adults but these same AEDs have limited efficacy in the neonatal brain. Despite evidence that the AED phenobarbital is effective in only 30-50% of babies, it remains the standard first-line treatment for neonatal seizures in neonatal intensive care units around the world.In the perinatal hypoxic-ischaemic (HI) brain, over activation of excitatory neurotransmitter systems plays a key role in the generation of seizures and excitotoxic neuronal cell injury and cell death. In the mature brain GABA (γ-aminobutyric acid) hyperpolarises neurones and inhibits neuronal firing, thus providing a protective mechanism by reducing excitability.However, in the immature brain GABA depolarises the neurone due to differences in the chloride (Cl -) gradient across the membrane, and instead creates excitation when GABA binds to the GABAA receptor. Understanding the physiological consequences of activation of the GABA system in the HI newborn brain and in the presence of seizures, is critical to identifying the mechanisms behind the apparent failure of AEDs in the newborn as well as driving development of appropriate drugs for the treatment of neonatal seizures.The general aim of this thesis was to investigate changes to the GABAergic system following perinatal hypoxia and seizures, by examining alterations in expression of the GABAA receptor (GABAAR) α-subunit in the pig brain. The piglet brain growth trajectory most closely mirrors that of the human neonate, with respect to timing of myelination, neuronal and glial growth spurts, and the proportion of grey to white matter. Normal developmental expression of the α-subunits α1, α2, and α3 was characterised across gestation and multiple brain regions revealing the crossover in expression of α3 to α1 expression corresponding to the transition from foetal to postnatal life. There was a significant peak in α3 expression observed at 100d gestation (87% gestation), that coincides with a previously reported peak in prenatal pig brain growth.ii It has been suggested that the transition of...

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The Design of a Quantitative Western Blot Experiment

Cited by 296 publications

References 23 publications

Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

The effect of model rescaling and normalization on sensitivity analysis on an example of a MAPK pathway model

Examining the GABA<sub>A</sub> receptor alpha-subunits in the neonatal pig brain: changes across development and the effect of seizures after hypoxic-ischaemic brain injury

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