A systematic approach to quantitative Western blot analysis

Pillai-Kastoori, Lakshmi; Schutz-Geschwender, Amy; Harford, Jeff A.

doi:10.1016/j.ab.2020.113608

Cited by 240 publications

(154 citation statements)

References 94 publications

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“…Immunoblotting was performed as previously described ( Li et al, 2019 ; Pillai-Kastoori et al, 2020a , b ). In brief, 20–30 μg protein samples were separated by SDS-PAGE on 12% polyacrylamide gels by electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways

Wen

Wang

et al. 2020

Front. Mol. Neurosci.

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Neurite outgrowth is essential for brain development and the recovery of brain injury and neurodegenerative diseases. In this study, we examined the role of the neurotrophic factor MANF in regulating neurite outgrowth. We generated MANF knockout (KO) neuro2a (N2a) cell lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and demonstrated that MANF KO N2a cells failed to grow neurites in response to RA stimulation. Using MANF siRNA, this finding was confirmed in human SH-SY5Y neuronal cell line. Nevertheless, MANF overexpression by adenovirus transduction or addition of MANF into culture media facilitated the growth of longer neurites in RA-treated N2a cells. MANF deficiency resulted in inhibition of Akt, Erk, mTOR, and P70S6, and impaired protein synthesis. MANF overexpression on the other hand facilitated the growth of longer neurites by activating Akt, Erk, mTOR, and P70S6. Pharmacological blockade of Akt, Erk or mTOR eliminated the promoting effect of MANF on neurite outgrowth. These findings suggest that MANF positively regulated neurite outgrowth by activating Akt/mTOR and Erk/mTOR signaling pathways.

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Section: Methodsmentioning

confidence: 99%

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways

Wen

Wang

et al. 2020

Front. Mol. Neurosci.

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“…The minimal manipulation of cells in CLIC helped to yield faster and more reproducible results compared to our previous experience with Western blots and dot blots, likely because CLIC does not use several steps that can adversely affect reproducibility 8 , 31 . Specifically, the cell lysis step in Western blotting releases many proteases and phosphatases that can degrade the protein of interest over time, requiring the use of protease inhibitor cocktails and cold temperatures 32 , 33 .…”

Section: Discussionmentioning

confidence: 95%

Using chemiluminescence imaging of cells (CLIC) for relative protein quantification

Fisher

Sørensen

Abu-Humaidan

2020

Sci Rep

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Cell physiology and cellular responses to external stimuli are partly controlled through protein binding, localization, and expression level. Thus, quantification of these processes is pivotal in understanding cellular biology and disease pathophysiology. However, it can be methodologically challenging. Immunofluorescence is a powerful technique, yet quantification by this method can be hampered by auto-fluorescence. Here we describe a simple, sensitive and robust chemiluminescence-based immunoassay (chemiluminescence imaging of cells; CLIC) for relative quantification of proteins. We first employed this method to quantify complement activation in cultured mammalian cells, and to quantify membrane protein expression, shedding, binding and internalization. Moreover, through specific membrane permeabilization we were able to quantify both cytosolic and nuclear proteins, and their translocation. We validated the CLIC quantification method by performing parallel experiments with other quantification methods like ELISA, qPCR, and immunofluorescence microscopy. The workflow of the immunoassay was found to be advantageous in certain instances when compared to these quantification methods. Since the reagents used for CLIC are common to other immunoassays with no need for specialized equipment, and due to the good linearity, dynamic range and signal stability inherent to chemiluminescence, we suggest that this assay is suitable for both small scale and high throughput relative protein quantification studies in whole cells.

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“…In conclusion, UVHis-PAGE can be an ideal tool for the rapid and straightforward detection of His-tagged proteins in applications where specialized fluorescence detection is unavailable or traditional antibody-based immunoblotting is too costly or time-consuming. Apart from indicating the presence of a particular epitope, immunoblotting with secondary antibodies, based on both chemiluminescent ( 28 , 29 ) and fluorescent ( 28 , 30 , 31 ) detection, has been used as a quantitative tool to determine the epitope amount. For a given set of conditions, such as gel percentage and composition or blotting membrane composition, the type and concentration of Ni 2+ -MCH conjugate used, the staining and destaining times, and the imaging parameters, we envision that our methods can also be used as quantitative tools, mainly through the use of a calibration curve similar to the dependence described in Fig.…”

Section: Discussionmentioning

confidence: 99%

Simplified detection of polyhistidine-tagged proteins in gels and membranes using a UV-excitable dye and a multiple chelator head pair

Raducanu

Isaioglou

Raducanu

et al. 2020

Journal of Biological Chemistry

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The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion–loaded N-nitrilotriacetic acid–based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion–loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion–loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.

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A systematic approach to quantitative Western blot analysis

Cited by 240 publications

References 94 publications

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways

Using chemiluminescence imaging of cells (CLIC) for relative protein quantification

Simplified detection of polyhistidine-tagged proteins in gels and membranes using a UV-excitable dye and a multiple chelator head pair

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