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Glomelt thermal shift protein stability kit

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The GloMelt Thermal Shift Protein Stability Kit is a laboratory tool designed to assess the thermal stability of proteins. It utilizes a fluorescent dye that binds to hydrophobic regions of proteins, allowing for the detection of protein unfolding as the temperature is increased.

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Lab products found in correlation

Rotor gene q qpcr instrument, by Qiagen (1 mentions)

6 protocols using glomelt thermal shift protein stability kit

1

Thermal Stability of nanoCLAMPs

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The melting temperature of purified nanoCLAMPs was determined using GloMelt Thermal Shift Protein Stability Kit (Biotium) per the manufacturer’s instructions. Briefly, purified nanoCLAMPs were adjusted to 1 mg/ml in MBS, 1 mM CaCl2, pH 6.5 and diluted in half with 2× GloMelt (Biotium) and aliquoted to 386-well plate and sealed with optical film. The plate was then heated in a Quantstudio five qPCR machine using SYBR Green reporter with no passive reference. The heating profile was 25 °C for 2 min; ramp at 0.05 °C/sec to 99 °C; 99 °C for 2 min. Tm is defined as the inflection point in the unfolding curve.
Suderman R.J., Gibson S.D., Strecker M., Bonner A.M, & Chao D.M. (2023). Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands. The Journal of Biological Chemistry, 299(7), 104910.
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2

Thermal Stability Evaluation of SarA

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The GloMelt Thermal Shift Protein Stability Kit (Biotium) was used to determine the thermal stability of SarA with or without telithromycin. A 20-µL reaction mixture was prepared in buffer (25 mM Tris, pH 7.4; 100 mM NaCl; and 0.1% NP40) containing 200 µM SarA, 1× GloMelt dye, and 4 mM telithromycin. The control reaction contained the same concentration of DMSO without telithromycin. Reaction mixtures were analyzed in a thermocycler (QuantStudio 6 Pro) using melt curve settings as follows: initial hold: 25°C, melt curve temp: 25°C–99°C, and ramp rate: 0.05°C/s. Data acquisition was done using a SYBR Green filter set with excitation and emission wavelengths of 470 and 520 nm, respectively. Analysis of Tm was performed using the Boltzmann equation from a plot of fluorescence intensity vs temperature in OriginPro software.
Campbell M.J., Beenken K.E., Spencer H.J., Jayana B., Hester H., Sahukhal G.S., Elasri M.O, & Smeltzer M.S. (2023). Comparative evaluation of small molecules reported to be inhibitors of Staphylococcus aureus biofilm formation. Microbiology Spectrum, 12(1), e03147-23.
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3

Thermal Stability Profiling of TgLEA8x0 Protein

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Differential scanning fluorimetry was performed using the GloMelt thermal shift protein stability kit (Biotium, USA). Master mixes containing 9 μL of the GloMelt working solution (prepared according to the manufacturer’s instructions) and TgLEA8x0 at a final concentration of 15 μM, or the IgG control included with the kit at the proposed final concentration of 0.5 μg/mL, were prepared on ice and the volume adjusted to 90 μL with HEPES buffer. Four 20-μL replicates were transferred to a 96-well qPCR plate, covered with corresponding strips and kept on ice. The plate was quickly spun down for 1 min at 200 × g and immediately transferred to a CFX96 Touch Real-Time PCR reader (Bio-Rad) for analysis. The following melting curve parameters were applied: preincubation = 2 min at 0°C; range = 0 to 99°C; increments = 0.5°C/min; read interval = 1 min. Data were exported to .txt format and analyzed using the R package ggplot2 0.9.0 and the stat_smooth function.
Arranz-Solís D., Warschkau D., Fabian B.T., Seeber F, & Saeij J.P. (2023). Late Embryogenesis Abundant Proteins Contribute to the Resistance of Toxoplasma gondii Oocysts against Environmental Stresses. mBio, 14(2), e02868-22.
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4

Thermal Stability of NSP14/10 Complex

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Melting temperature of the NSP14/10 complex in the presence or absence of select compounds were assayed by differential scanning fluorimetry using either Protein Thermal Shift™ (Thermo Scientific) or GloMelt™ Thermal Shift Protein Stability Kit (Biotium) according to the manufacturer’s protocol. Assays were performed in a 96-well qPCR plate in a final volume of 25 μL in the following reaction buffer: 50 mM TRIS-HCl pH=7.5, 2 mM MgCl2, 2 mM DTT. NSP14 and NSP10 were allowed to pre-complex at an equimolar ratio of 5 μM at RT for 15 min before adding the compounds at 50 μM final concentration. The temperature was linearly increased in a QuantStudio3 Real-Time PCR System with a step of 0.05 °C/s, from 25 °C to 95 °C, and fluorescence readings were taken at each interval (Protein Thermal Shift Assay: ROX filter set; GloMelt Thermal Shift Assay: FAM filter set). Melting temperatures were calculated as the inflection point of the melting curve using the derivative analysis function of the QuantStudioTM Design & Analysis Software (version 1.4.3).
Rona G., Zeke A., Miwatani-Minter B., de Vries M., Kaur R., Schinlever A., Garcia S.F., Goldberg H.V., Wang H., Hinds T.R., Bailly F., Zheng N., Cotelle P., Desmaële D., Landau N.R., Dittmann M, & Pagano M. (2021). The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target. Cell Death and Differentiation, 29(2), 285-292.
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5

Protein-Ligand Binding Analysis by CETSA

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The protein-ligand binding was first studied by cellular thermal shift assay (CETSA) by following previously described protocol [22 (link)]. Briefly, we treated the cells (3×106) with ASR490, incubated at different temperatures (38–55°C) to denature and precipitate proteins, performed cell lysis and centrifuged at 13000g for 10 min to collect the soluble fraction. Equal amount of cell lysate was used for ELISA with NRR antibody (Cat: NBP2–62557; Novus Biologicals). GloMelt Thermal Shift Protein Stability assay was performed as per the kit instructions (GloMelt™ Thermal Shift Protein Stability Kit; Biotium; Fermont, CA). Briefly, a qPCR reaction was setup with the purified NRR protein (Origene technologies: TP606288), the GloMelt fluorescent dye and ASR490 (10μg per reaction). A protein melt run profile was generated and Tm (melting temperature) was calculated using DNA melt curve software. To analyze protein melting, the Tm was considered at the lowest -dF/dT value (at the lowest point on the curve).
Santana M., Pereira M.M., Elias N.P., Soares C.M, & Teixeira M. (2001). Gene Cluster of Rhodothermus marinus High-Potential Iron-Sulfur Protein:Oxygen Oxidoreductase, a caa3-Type Oxidase Belonging to the Superfamily of Heme-Copper Oxidases. Journal of Bacteriology, 183(2), 687-699.
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6

Thermal Stability Analysis of SAMHD1

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Thermal melt measurements were made using the GloMelt Thermal Shift protein stability kit from Biotium on a Qiagen Rotor-gene Q qPCR instrument. Solutions of 50 mM HEPES—pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.5 mM TCEP, 3 μM SAMHD1, 1X GloMelt dye and 0/2 mM dGTPαS were subjected to a temperature gradient from 25 to 85°C at 0.5°C intervals. Each step in the gradient was held for three seconds and the fluorescence of the GloMelt dye was measured Rotor-gene Q green channel. Automatic gain optimization was performed all of the samples before execution of the temperature gradient with a signal limit of seven. Three replicates were recorded for each measured condition. Melt temperatures for each replicate were calculated using the program TSA-CRAFT (Supplemental Table S2) (29 (link)). To display differences in melt temperature between different SAMHD1 constructs, averaged traces were differentiated and normalized in Prism using 6th order polynomial smoothing considering five neighbors on each side.
Orris B., Huynh K.W., Ammirati M., Han S., Bolaños B., Carmody J., Petroski M.D., Bosbach B., Shields D.J, & Stivers J.T. (2022). Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation and ssDNA binding kinetics. Nucleic Acids Research, 50(13), 7545-7559.
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