Rapid quantification of total protein with surface‐enhanced Raman spectroscopy using <i>o</i>‐phthalaldehyde

Eryılmaz, Merve; Zengi̇n, Adem; Boyaci, İsmail Hakkı; Tamer, Uğur

doi:10.1002/jrs.5099

Cited by 7 publications

(7 citation statements)

References 36 publications

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“…We chose this signal as internal standard to measure the protein concentration because of its insensitivity to secondary structure, strong signal, and direct correlation to the phenylalanine count. [35][36][37][38] The procedure was validated by measuring a solution at known concentration of bovine serum albumin (BSA) and normalizing the maximum intensity of the β-Phe peak with the phenylalanine count in the BSA sequence ( Figure 1F). Next, we applied this method to measure the concentration of chimeric protein inside the condensed phase by focusing the laser beam on the centre of the droplets ( Figure 1G).…”

Section: Resultsmentioning

confidence: 99%

Acceleration of an Enzymatic Reaction in Liquid Phase Separated Compartments Based on Intrinsically Disordered Protein Domains

et al. 2020

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Spontaneous liquid demixing of biomolecules appears to be an efficient strategy developed by cells to organize reactions in space and time. This process allows cells to modulate biochemical reactions by locally changing the concentration and the environment of specific components. Here, we develop a strategy to couple the formation of biomolecular liquid compartments with reactions occuring within them. In particular, we conjugate a kinase enzyme with biologically derived low complexity domains and develop synthetic micro-reactors that locally increase the enzyme concentration up to 140-fold. We show that these micro-reactors are characterized by a polarity comparable to methanol which promotes recruitment of small molecules. Despite exhibiting higher viscosity with respect to the surrounding solution, the reactors are liquid-like and allow molecular diffusion within their interior. We demonstrate that the local increase in enzyme concentration accelerates the corresponding enzymatic rate up to 5-fold. This flexible strategy enables the generation of biomolecular microreactors with enhanced reactivity, with potential applications in heterogeneous biocatalysis.

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Section: Resultsmentioning

confidence: 99%

Acceleration of an Enzymatic Reaction in Liquid Phase Separated Compartments Based on Intrinsically Disordered Protein Domains

et al. 2020

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“…FT-Raman spectroscopy is an efficient technique for the investigation of structural changes of protein molecules (Liang et al, 2006). Eryilmaz et al (2017) used surface-enhanced Raman spectroscopy (SERS) for analysis of skimmed milk. SERS gives enhanced Raman signal from an analyte molecule which was adsorbed on metal surfaces.…”

Section: Routine Methodsmentioning

confidence: 99%

Milk Protein Analysis: an Overview of the Methods - Development and Application

Kala

Samková

Hanuš³

et al. 2019

Acta Univ. Agric. Silvic. Mendelianae Brun.

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Milk protein content is an important component of milk, especially from a nutritional point of view and also for payment purposes. The aim of work was to draw up an overview on reference and routine methods for protein determination. Reference methods perform accurate analyses comply according to the International Standard ISO whereas routine methods perform analyses using routine instrumental techniques for faster and cheaper results with acceptable accuracy and a large number of processed samples. In most of cases, using of routine indirect methods for milk protein analysis requires their specific calibrations according to biological kind of measured milk (cow’s, goat’s or sheep’s milk) or specific conditions of milk technology treatment. Also, the quality control measures have a significant role for result determination reliability.

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“…This method is sensitive and structural selective, “it can provide unique insights into protein dynamics” and is able to investigate slow changes in protein conformation (Balakrishnan et al 2008). Traditional Raman spectroscopy needs a sample concentration of more than 1 g/l (Wen 2007), whereas surface enhanced Raman spectroscopy (SERS) is more sensitive (> 0.08 g/l) (Eryilmaz et al 2017). …”

Section: Monitoring Toolsmentioning

confidence: 99%

Wanted: more monitoring and control during inclusion body processing

Humer

Spadiut

2018

World J Microbiol Biotechnol

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Inclusion bodies (IBs) are insoluble aggregates of misfolded protein in Escherichia coli. Against the outdated belief that the production of IBs should be avoided during recombinant protein production, quite a number of recombinant products are currently produced as IBs, which are then processed to give correctly folded and soluble product. However, this processing is quite cumbersome comprising IB wash, IB solubilization and refolding. To date, IB processing often happens rather uncontrolled and relies on empiricism rather than sound process understanding. In this mini review we describe current efforts to introduce more monitoring and control in IB processes, focusing on the refolding step, and thus generate process understanding and knowledge.

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Rapid quantification of total protein with surface‐enhanced Raman spectroscopy using o‐phthalaldehyde

Cited by 7 publications

References 36 publications

Acceleration of an Enzymatic Reaction in Liquid Phase Separated Compartments Based on Intrinsically Disordered Protein Domains

Acceleration of an Enzymatic Reaction in Liquid Phase Separated Compartments Based on Intrinsically Disordered Protein Domains

Milk Protein Analysis: an Overview of the Methods - Development and Application

Wanted: more monitoring and control during inclusion body processing

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