Common Pitfalls and Recommendations for Using a Turbidity Assay to Study Protein Phase Separation

Huang, Yanan; Bai, Yinge; Jin, Wenhan; Shen, Di; Lyu, Haochen; Zeng, Lianggang; Wang, Mengdie; Liu, Yu

doi:10.1021/acs.biochem.1c00386

Cited by 9 publications

(4 citation statements)

References 66 publications

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“…An increase in the mixed ratio would lead to the segregated phase separation, which results in the formation of the AuNR−H-SA−CTAB network (discussed later) deposited as thin films. A simple route to understanding the kinetics of the development of insoluble aggregated proteins is provided, in particular, by time-dependent studies of turbidity 72 (Figure 7C,D) by monitoring the optical density value at 330 nm (OD 330 ) with time. Two experimental sets are prepared: one in which four different concentrations of HSA are added one by one in a solution of AuNRs with a fixed concentration and another in which four different concentrations of AuNRs are added one by one in a solution of HSA with a fixed concentration.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Thin Film Formation of HSA in the Presence of CTAB-Capped Gold Nanorods through Phase Separation

Halder,

Sabnam,

Das

et al. 2024

Langmuir

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Phase behavior in protein−nanoparticle systems in light of protein corona formation has been investigated. We report the formation of HSA thin films following the addition of a solid protein to a solution of CTAB-capped gold nanorods (AuNRs) via phase separation. The phase separation behavior was observed through UV−vis spectroscopy, turbidity assays, and DLS studies. UV−vis spectra for the protein-AuNR solution indicated a possible self-assembly formation by CTAB−HSA complexes and AuNR−HSA conjugates. The turbidity was found to increase linearly up to 30−50% v/v for each component. The growth phase slope is proportional to the concentration of the components, AuNRs, and HSA, with no lag phase. Dynamic light scattering (DLS) shows the formation of larger aggregates with time, implying a segregated phase of AuNR−HSA and a CTAB−HSA−AuNR network. ζ-potential values confirm surface modification, implying protein corona formation on nanorods. The thin films were also characterized using SEM, AFM, SAXS, XPS, FTIR, and TGA studies. SEM images show a smooth surface with a reduced number of pores, indicating the compactness of the deposited structure. AFM shows two different structural pattern formations with the deposition, indicating possible self-assembly of the protein-conjugated nanoparticles. FTIR studies indicate a change in the hydrogen bonding network and confirm the CTAB−HSA−AuNR complex network formation. The XPS studies indicate Au−S bond formation, along with Au−S−S−Au interactions. SAXS studies indicate the formation of aggregates (oligomers), as well as the presence of dominant attractive intermolecular interactions in the thin films.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Thin Film Formation of HSA in the Presence of CTAB-Capped Gold Nanorods through Phase Separation

Halder,

Sabnam,

Das

et al. 2024

Langmuir

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“…80 Protein aggregation was scanned using UV-Vis spectrophotometry, the detection limits and linearity of turbidity detection were shown to be improved by simple sample dilution, and the highly dynamic and reversible LLPS required direct fluorescence imaging. 12 Longnecker 81 et al provided a reliable method called the straw technique to demonstrate the occurrence of LLPS in organic brine systems. The dynamics and interactions between dynamics of protein solution LLPS were investigated using X-ray spectroscopy.…”

Section: Comparison Of Different Detection Methodsmentioning

confidence: 99%

“…Various detection methods have developed rapidly (Fig. 1b), for instance fluorescence imaging techniques, 8,9 functional microscope imaging techniques, 10,11 turbidity monitoring, 12 macromolecule congestion sensing, 13 in silico analysis, 14 etc. However, these methods involve multiple fields, it is difficult for researchers to take advantage of each method.…”

Section: Introductionmentioning

confidence: 99%

Fluorogenic methodology for visualization of phase separation in chemical biology

Fang

Huang

et al. 2023

Org. Biomol. Chem.

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“…Turbidity leads to a decrease in the intensity of the light that passes through a turbid solution due to light scattering. Turbidity measurements of HI (μM) incubated without and with rivastigmine in an increasing manner were carried out using an absorption spectrometer (Hitachi 2900) at a fixed wavelength of 350 nm in a cuvette of 1 cm path length (Zhao et al, 2016;Huang et al, 2021).…”

Section: Turbiditymentioning

confidence: 99%

Unraveling the Interaction of a Neurological Drug Rivastigmine with Human Insulin Protein: A Biophysical Method in Combination with Molecular Docking and Molecular Dynamics Simulation

Aljarba,

Alqahtani,

Alkahtani

et al. 2023

Journal of Disability Research

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The study aims to examine the aggregation of proteins due to their association with various conformational problems including well-known neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. In this context, we have studied the mechanistic route of the neurological drug rivastigmine on human insulin (HI) protein aggregation. The Thioflavin T and light scattering studies establish that the drug has the ability to inhibit HI fibrillation. It is pertinent to note here that rivastigmine has been anticipated to cross the blood–brain barrier. The dye 8-anilino-1-naphthalenesulfonic acid binding assays exhibit that the drug interferes with the hydrophobic domain of the protein because of intermolecular interaction. Furthermore, molecular docking and molecular dynamics simulation studies revealed the interactions of rivastigmine at the receptor site of acetylcholinesterase (AChE) and Aβ-42, thereby inhibiting the hydrolytic activity. Rivastigmine forms a hydrogen bond with the Tyr337 residue of AChE and His14 (chain C) and Gly33 (chain A) residues of Aβ-42, in addition to several weak interactions. The results showed the fundamental mechanisms of the inhibition of HI fibrillation by neurological drugs, which may provide ideas about the rational design of new lead therapeutics against amyloidosis.

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Common Pitfalls and Recommendations for Using a Turbidity Assay to Study Protein Phase Separation

Cited by 9 publications

References 66 publications

Thin Film Formation of HSA in the Presence of CTAB-Capped Gold Nanorods through Phase Separation

Thin Film Formation of HSA in the Presence of CTAB-Capped Gold Nanorods through Phase Separation

Fluorogenic methodology for visualization of phase separation in chemical biology

Unraveling the Interaction of a Neurological Drug Rivastigmine with Human Insulin Protein: A Biophysical Method in Combination with Molecular Docking and Molecular Dynamics Simulation

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