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Floating sphere assay: A rapid qualitative method for microvolume analysis of gelation.

ABSTRACT: A huge, unprecedented demand for gelatin coupled with its implications on global sustainability has resulted in the need to discover novel proteins with gelling attributes for applications in the food industry. Currently used gelation assays require large sample volumes and thus the screening for novel gelling proteins is a formidable technical challenge. In this paper, we report the 'Floating Sphere Assay' which is a simple, economical, and miniaturized assay to detect minimum gelling concentration with volumes as low as 50 μl. Results from the Floating Sphere Assay are consistent with currently used methods for gelation tests and accurately estimate the Minimum Gelling Concentrations (MGCs) of gelatin, κ-carrageenan and gellan gum. The assay was also able to differentiate the strengths of strong and weak gellan gum gels prepared at pH 3.5 and pH 7.0 respectively. The Floating Sphere Assay can be utilized in high-throughput screens for gelling proteins and can accelerate the discovery of gelatin substitutes.

SUBMITTER: Tay UJ

PROVIDER: S-EPMC9269766 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We present here a modified, sonication-assisted transient transformation assay for rapid analysis of cis-regulatory elements. We tested promoter elements from MIR159B locus of Brassica juncea by generating stable transgenic lines and compared the transcriptional activity of GUS reporter with that of the transient assay method. To obtain reliable and repeatable results, and to omit false-positive data, we optimized several parameters including sonication duration and cycle and concentration of Agrobacterium tumefaciens measured as optical density (O.D.) at 600 nm. To the best of our knowledge, this is the first report of promoter characterization of MIR159B from Brassica juncea, and comparative analysis of stable and transient lines. Our analysis shows that the protocol described herein allows understanding promoter activity/transcriptional control in tissues other than leaf or protoplast which have remained the mainstay for transient analysis thus far. We tested reporter gene GUS under the control of constitutive promoter, CaMV 35S, and MIR159b from Brassica juncea. We optimized the duration of sonication (5-, 10- and 15-min cycle), bacterial density (measured as O.D at 600 nm = 0.6/0.8/1.0) and Agro-infection time (5, 10, 15 min), and co-cultivation (12-, and 24-h). Sonication cycle of 10-min, followed by Agro-infection and co-cultivation with Agrobacterium tumefaciens with O.D. 600 nm = 0.8 and for 12 h was found to be optimum. We could successfully express reporter genes in deep-seated tissues such as floral organs and pollen grains where it was previously not possible to perform transient assay. Constitutive GUS activity was observed when reporter was placed under control of the constitutive promoter of CaMV 35S. Reporter GUS when placed under transcriptional control of MIR159b promoter from Brassica juncea showed reporter activity in floral tissues, in mature pollen grains. Comparative analysis of reporter activity from stable transgenic lines at T2 generation with that of transient assay system reveals identical to near-identical reporter activity. Transient assay could be successfully performed in tissues collected not only from Arabidopsis thaliana, but also from Brassica juncea and Brassica nigra to demonstrate its wide applicability. Our modified method thus has the potential of quick and rapid analysis of promoter activity and allows us to record the developmental dynamics and spatio-temporal expression pattern driven by specific promoters. Suitable modification and controls should also allow analysis of hormonal regulation and identification of trans-factors via DNA-protein interactions. Furthermore, this method can also be extended to study promoters under various environmental conditions that otherwise do not allow growth and complete life cycle of healthy plants and can be modified to test reporter activity in other non-model plants or plants with long life cycle.

Project description:PurposeTo develop a microvolume analytical method for measurement of the aflibercept concentration in human intraocular fluid and plasma.MethodsWe analyzed trace amounts of aflibercept in human aqueous humor using Fab-selective proteolysis and nano-surface and molecular-orientation limited (nSMOL) proteolysis, coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Patients with age-related macular degeneration or diabetic macular edema were recruited. Just after an injection of 50 µL of aflibercept, regurgitate from needle holes was collected with a micropipette pressed to the side of the injection hole within 10 seconds. The median amount of regurgitate was 4 µL (range, 1-18 µL).ResultsIn human plasma, the aflibercept concentration ranged between 0.195 and 50 µg/mL when using the quantitative signature peptide IIWDSR (aa. 56-61) present on the vascular endothelial growth factor receptor 1 domain of aflibercept. The method was validated by evaluating its linearity, carryover, selectivity, accuracy and precision, dilution effect, and sample/processing stability. As only a minimal amount of regurgitate through needle holes can be sampled, we performed and verified the aflibercept assay using patient samples after 1:10 dilution with control human plasma, a recognized diluent. The median concentration of aflibercept in the regurgitate was 240 µg/mL (range, 13-4300 µg/mL).ConclusionsOur findings indicate that the aflibercept assay using human intraocular fluid can be reliably performed using nSMOL coupled with LC-MS/MS.Translational relevanceThis technique for quantifying aflibercept in the regurgitate suggests that the amount of drug lost post-injection can be ignored, even in patients with a relatively large leak after vitreous injection. This new methodology suggests possible therapeutic responses and may be employed as a general analytical method for trapping many biologics, such as vascular endothelial growth factor, in various types of clinical samples, unaffected by proteinaceous or small organic pharmaceuticals.

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Floating sphere assay: A rapid qualitative method for microvolume analysis of gelation.

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