5 bromo 4 chloro 3 indolyl β d galactopyranoside x gal solution  (Thermo Fisher)

Journal: Cell Discovery

Article Title: TGM2-mediated serotonylation of GPX4 confers ferroptosis resistance to promote gastric tumorigenesis

doi: 10.1038/s41421-026-00885-6

Figure Lengend Snippet: a TGM2-dependent transamidation signals in recombinant GPX4 (monitored via monodansylcadaverine fluorescence) that were abolished by either cystamine inhibition or 5-HT competition. b Schematic illustration of the TGM2-dependent transamidation of glutamine residues in substrate proteins. c MS/MS spectra of the serotonylated GPX4 peptide (GPX4ser: Q55, Q77) derived from GPX4 (in vitro). The b ion refers to the N-terminal portion of the peptide, and the y ion indicates to the C-terminal portion. d SILAC experimental workflow for GPX4 serotonylation detection by LC-MS/MS using 5-HT and 5-HT- d 4 . TGM2 catalyzes the transamidation of glutamine residues in the GPX4 protein. e Chromatographic overlay of 5-HT and 5-HT- d 4 , demonstrating nearly identical retention times or 45.03 min and 45.05 min, respectively (top). MS analysis of 5-HT and 5-HT- d 4 revealed distinct isotopic peaks. Mass spectra of Light, with m/z of 727.36 and Heavy, with m/z of 729.37 m/z for 5-HT and 5-HT- d 4 , respectively, illustrating the clear separation between the light and heavy isotopic labels and confirming the use of deuterium-labeled serotonin for dual validation (bottom). f Tandem MS/MS spectrum of the peak at m/z 727.36 (5-HT Light) showing the fragmentation pattern of GPX4 at the serotonylated site, confirming the presence of 5-HT modification (top). Tandem MS/MS spectrum of the peak at m/z 729.37 (5-HT- d 4 Heavy) showing the corresponding fragmentation pattern, confirming the site-specific serotonylation of GPX4 (bottom). g Immunoblot and streptavidin pull-down analysis of Flag-GPX4 with various mutations (Q54A, Q77A, and Q2A) in HEK293T cells treated with 5-PT, confirming the loss of serotonylation at specific sites. Quantification is shown on the right (mean ± SEM; ordinary one-way ANOVA, n = 3).

Article Snippet: Each 50 μL reaction mixture contained 1× 50 mM HEPES reaction buffer (1× protease inhibitor), 5 mM 5-HT- d 4 (MCE, HY-B1473S) or 5 mM 5-HT combined with 5 mM CaCl 2 , 1.25 μg TGM2, and GPX4 recombinant protein (50 μg) and was established on ice.

Techniques: Recombinant, Fluorescence, Inhibition, Tandem Mass Spectroscopy, Derivative Assay, In Vitro, Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Labeling, Biomarker Discovery, Modification, Western Blot

Journal: Food Chemistry: Molecular Sciences

Article Title: Usage of nanobody-beta-galactosidase fusion in immunoassays and its application in detecting a peanut allergen

doi: 10.1016/j.fochms.2026.100357

Figure Lengend Snippet: β-Gal detection of the association between its fusion partner Nb16 and Ara h 3 by ELISA. The utility of β-gal as a colorimetric enzyme in ELISA was assessed. Nb16-βgal association with Ara h 3 coated on the surface of the wells, but not with the control proteins, was detected. (A) SDS-PAGE analysis of Ara h 3 and control proteins used to coat the microplate. Nonreduced (lane 1) and reduced (lane 2) Ara h 3 were separated on a 4–12% SDS gel and stained with CBB. Chicken allergen Gal d 2 (lane 3) and cow's milk allergen Bos d 4 (lane 4) were included as control samples. The molecular masses (in kDa) of the proteins in the marker (lane M) are shown on the right side of the gel images. (B) The kinetic curves of the signal readout during plate incubation after the β-gal substrate ONPG was added. The black line shows the average signal of the wells incubated with TBS during the coating step. Red, green, and blue lines show the average signals of the wells coated with Gal d 2, Bos d 4, and Ara h 3, respectively. All the coating samples were at a concentration of 20 μg/mL. (C) A bar representation of β-gal detection in the ELISA experiment using the endpoint data. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cow's milk allergen Bos d 4 (alpha-lactalbumin) was provided by Hilmar Cheese Company (Hilmar, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control, SDS Page, SDS-Gel, Staining, Marker, Incubation, Concentration Assay

Journal: Food Chemistry: Molecular Sciences

Article Title: Usage of nanobody-beta-galactosidase fusion in immunoassays and its application in detecting a peanut allergen

doi: 10.1016/j.fochms.2026.100357

Figure Lengend Snippet: Detection specificity of Nb16 by immunoblot. The utility of β-gal as an enzyme for colorimetric detection in immunoblot was assessed. Black circles were drawn with a permanent marker to provide coordinates for spotting proteins onto the membrane. Control sample Bos d 4 was spotted at rows 2 and 3 (counting from the top) in the first column (counting from the left). Ara h 3 was spotted at rows 2 and 3 of the second column. Ara h 3 was also spotted at columns 3 and 4, with ½ and ¼ of the sample volume spotted at column 2.

Article Snippet: Cow's milk allergen Bos d 4 (alpha-lactalbumin) was provided by Hilmar Cheese Company (Hilmar, CA, USA).

Techniques: Western Blot, Marker, Membrane, Control

Journal: bioRxiv

Article Title: Selective Hydrolytic Defluorination of Branched Perfluorooctanoic Acid Isomers by a Haloacid Dehalogenase

doi: 10.64898/2026.04.19.719434

Figure Lengend Snippet: (a) Docking model showing PFOA bound within the active-site pocket of 4A. (b) Enlarged view of the PFOA-binding site, illustrating predicted hydrogen-bonding interactions between the PFOA carboxylate group and surrounding active-site residues. (c) Two-dimensional interaction map of the PFOA–4A complex, highlighting hydrogen-bonding interactions involving the carboxylate group (green dashed lines) and hydrophobic or polar contacts between PFOA and active-site residues. (d) Biosensor-based plate assay detecting fluoride release following high-concentration, scaled-up reactions. PFOA (0.5 mM) was incubated with purified 4A (500 µM) at 20℃ for 120 h prior to analysis. Following incubation, aliquots from the reaction mixture were analyzed using a fluoride-responsive riboswitch biosensor with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) as the chromogenic substrate. Enzyme-only, substrate-only, buffer-only controls, and fluoride standards (10 and 500 μM KF) are shown.

Article Snippet: 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) solution (20 mg/mL; Thermo Fisher Scientific, RO941) and 10 mM Tris buffer (pH 8.0, prepared from a 1 M stock; Thermo Fisher Scientific, AM9855G) were used for enzyme assays.

Techniques: Binding Assay, Concentration Assay, Incubation, Purification