Inactivation of Horseradish Peroxidase by Acid for Sequential Chemiluminescent Western Blot

Han, Shuting; Cui, Yan; Helbing, Dario-Lucas

doi:10.1002/biot.201900397

Cited by 12 publications

(15 citation statements)

References 15 publications

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“…Similar to many previous efforts to solve this issue [15][16][17][18], we tried to develop an HRPinactivating method, instead of stripping out the HRP-conjugated antibody. Meanwhile, we intended to avoid the use of currently known reagents, such as the chromogenic substrate, H2O2, NaN3, or AA, because of their respective drawbacks.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, H 2 O 2 has a slightly irritating odor, and appears to be an animal carcinogen and mutagen [19]. Under a pre-optimized, case-sensitive condition, AA is also able to inactivate HRP, but it may need further optimization on a case-by-case basis, and should be used with caution because it is a strong eye, skin, and mucous membrane irritant, with high volatility under the applicable condition [18].…”

Section: Introductionmentioning

confidence: 99%

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A simple, flexible and high-efficiency western blot analysis for age-related human induced neurons

Shen

Liu

Zhu³

et al. 2023

Preprint

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High-throughput western blot (WB) analysis of small and precious samples, including various age-related subtype-specific human induced neurons (hiNs), endows the ability to obtain more consistent, comparable, and informative data from materials with highly limited availability. In this study, p-toluenesulphonic acid (PTSA), an odorless tissue fixative, was used to inactive HRP for developing a high-throughput WB method. PTSA-treated blots showed fast and efficient inactivation of HRP without detectable protein loss and epitope damage. With a brief PTSA-treatment before every next probing, 10 proteins of dopaminergic hiNs could be sequentially, sensitively, and specifically detected in a blot. These WB data proved the age-associated and neuron-specific features of hiNs and further revealed a sharp reduction of two Parkinson disease-associated proteins, UCHL1 and GAP43, in the normal aging dopaminergic neurons. Together, this study developed a unique and high-efficiency WB analysis and pinpointed its special value for capturing robust useful data from limited, precious samples.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A simple, flexible and high-efficiency western blot analysis for age-related human induced neurons

Shen

Liu

Zhu³

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…In western blot experiments (Han et al, 2020), proteins were extracted from tissue samples in each group and quantified using the BCA Protein Assay Kit (Cat No. P0010S, Biyuntian, China).…”

Section: Western Blottingmentioning

confidence: 99%

Melatonin Regulates the Periodic Growth of Cashmere by Upregulating the Expression of Wnt10b and β-catenin in Inner Mongolia Cashmere Goats

Liu

et al. 2021

Front. Genet.

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Secondary hair follicle growth in cashmere goats has seasonal cycle changes, and melatonin (MT) has a regulatory effect on the cashmere growth cycle. In this study, the growth length of cashmere was measured by implanting MT in live cashmere goats. The results indicated that the continuous implantation of MT promoted cashmere to enter the anagen 2 months earlier and induce secondary hair follicle development. HE staining of skin tissues showed that the number of secondary hair follicles in the MT-implanted goats was significantly higher than that in the control goats (P < 0.05). Transcriptome sequencing of the skin tissue of cashmere goats was used to identify differentially expressed genes: 532 in February, 641 in October, and 305 in December. Fluorescence quantitative PCR and Western blotting results showed that MT had a significant effect on the expression of Wnt10b, β-catenin, and proteins in the skin tissue of Inner Mongolia cashmere goats. This finding suggested that MT alters the cycle of secondary hair follicle development by changing the expression of related genes. This research lays the foundation for further study on the mechanism by which MT regulates cashmere growth.

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“…The proteins were transferred onto the nitrocellulose membranes using Trans-Blot Turbo semi-dry transfer protocol (2.5A; 25V; 7 min). Protein fixation and residual peroxidase activity inhibition were done by incubation in 1% Ponceau S in 5% acetic acid [50]. Membranes were washed for 5 min in LSWB, blocked for 1 hour at room temperature (RT) in a blocking buffer (5% nonfat dry milk solution with 0,5% Tween 20 in low-salt washing buffer (LSWB: 10 mM Tris, 150 mM NaCl, pH 7.5)), and incubated with 1:500 anti-caspase-3 antibody (#9662S, Cell Signaling Technologies, USA) overnight at 4 ºC inbetween polyvinyl chloride (PVC) sheets to ensure equal reagent dispersion.…”

Section: Western Blotmentioning

confidence: 99%

Disbalance of the intestinal epithelial cell turnover and apoptosis in a rat model of sporadic Alzheimer’s disease

Homolak

Knezović

et al. 2021

Preprint

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BackgroundDyshomeostasis of the gastrointestinal (GI) system is investigated as a potential contributor to metabolic dysfunction, systemic and neuro-inflammation recognized as important pathophysiological drivers of neurodegeneration. Gastrointestinal redox dyshomeostasis and dysfunctional brain-gut incretin axis have been reported in the rat model of insulin-resistant brain state (IRBS)-driven neurodegeneration induced by intracerebroventricular administration of streptozotocin (STZ-icv). The aim was to assess i) whether GI oxidative stress is accompanied by structural and functional changes of the epithelial barrier; ii) whether the brain glucose-dependent insulinotropic polypeptide receptor (GIP-R) is also involved in redox regulation of the gut; and iii) whether the STZ-icv brain-gut axis is resistant to pharmacological inhibition of the brain GIP-R.MethodsForty three-month-old male Wistar rats were treated with 3mg/kg STZ-icv or vehicle. One month later the animals were randomized to receive either saline or 85 μg/kg GIP-R inhibitor [Pro3]-GIP intracerebroventricularly and sacrificed 30 minutes later. Thiobarbituric acid reactive substances (TBARS) were measured in plasma and duodenum. Duodenal sections were subjected to morphometric analysis. Caspase-3 expression and activation were analyzed by western blot and spatial signal analysis was done by multiplex fluorescent signal amplification (MFSA). Data were analyzed by linear and linear mixed modeling, and exploration was done by principal component analysis.ResultsInhibition of the brain GIP-R decreased plasma TBARS in the controls and the STZ-icv animals and increased duodenal TBARS only in the controls. Acute inhibition of brain GIP-R affects duodenal epithelial cell, but not villus structure, while all morphometric parameters were altered in the STZ-icv-treated animals. Morphometric changes in the STZ-icv animals were accompanied by reduced levels of activated and total regulator of apoptosis – caspase-3. Acute inhibition of brain GIP-R inactivated duodenal apoptosis at the level of caspase-3 activation.ConclusionsBrain GIP-R is involved in the regulation of the systemic and duodenal redox homeostasis and epithelial function. Duodenal oxidative stress in the STZ-icv rats is accompanied by the resistance of the brain-gut GIP axis and morphological changes indicative of abnormal epithelial cell turnover and dysfunctional GI barrier. Dysfunction of the brain-gut incretin axis might be an important etiopathogenetic factor in neurodegeneration and a potential pharmacological target.

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Inactivation of Horseradish Peroxidase by Acid for Sequential Chemiluminescent Western Blot

Cited by 12 publications

References 15 publications

A simple, flexible and high-efficiency western blot analysis for age-related human induced neurons

A simple, flexible and high-efficiency western blot analysis for age-related human induced neurons

Melatonin Regulates the Periodic Growth of Cashmere by Upregulating the Expression of Wnt10b and β-catenin in Inner Mongolia Cashmere Goats

Disbalance of the intestinal epithelial cell turnover and apoptosis in a rat model of sporadic Alzheimer’s disease

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