The TAMRA-VME probe assay was carried out as described previously [40 (link)]. Briefly, HEK293T cells transfected with USP19-CY-wt, USP19-CY-CA, USP19-ER-wt or USP19-ER-CS were lysed in TAMRA ABP buffer (50 mM Tris–HCl, pH 7.4, 250 mM sucrose, 5 mM MgCl2, 1 mM DTT, 0.5% zwitterionic surfactant CHAPS and 0.1% nonyl phenoxypolyethoxylethanol (NP40) supplemented with protease inhibitors. Then, the samples were sonicated for five cycles of 30 s (s) on and 30 s off on ice. Thereafter, the cell lysates were centrifuged at 16 × 103 g for 15 min at 4 °C, and the supernatants were transferred to fresh Eppendorf tubes to measure the protein concentrations. The carboxytetramethylrhodamine ubiquitin-vinyl methyl ester (TAMRA-Ub-VME) probe (UbiQ-050; UbiQ) was used at a concentration of 1 µM to label 25 µg of protein extracts in a total volume of 25 µL for 30 min at room temperature. The labeling reactions were terminated by the addition of sample buffer and heating to 100 °C for 10 min. The labeled proteins were separated by NuPAGE 4–12% Bis–Tris protein gels (WG1402BOX; Invitrogen), and the fluorescence signals were detected using the Typhoon FLA 9500 Molecular Imager (GE Healthcare) at an excitation wavelength of 550 nm and an emission wavelength of 590 nm.
Typhoon fla 9500 molecular imager
Manufactured by GE Healthcare
Sourced in United States
The Typhoon FLA 9500 Molecular Imager is a high-performance fluorescence and chemiluminescence imaging system designed for life science research and development. It provides sensitive detection and quantitative analysis of a wide range of fluorescent and chemiluminescent samples, including gels, membranes, and microplates.
Automatically generated - may contain errors
Lab products found in correlation
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Dcode universal mutation detection system, by Bio-Rad (1 mentions)
Sybr 1, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Dfc310 fx digital ccd color camera, by Leica (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
4 protocols using typhoon fla 9500 molecular imager
1
TAMRA-VME Probe Assay for USP19 Activity
2
Profiling Gut Bacterial 16S rDNA
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General bacterial 16S rDNA gene profiles of fecal bacteria in three model groups and control group were generated using the D-Code universal mutation detection system (Bio-Rad, Hercules, CA).Briefly, gels were prepared using 30–70% denaturing gradients containing 8% polyacrylamide. The amplification products of universal primers GC357F and 518 R targeting bacterial 16 S rDNA-V3 region were loaded onto the gel and electrophoresed in 1x Tris-acetate-EDTA buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) (TAE).Gels were run at 60 V for 2.5 h firstly, and then were run at 90 v for 12 h (DGGE-2001, C.B.S. Scientific, San Diego, CA, USA), and stained with SYBRI (Invitrogen, Carlsbad, CA, USA). Gel bands were then visualized with a Typhoon FLA 9500 Molecular Imager (GE Healthcare, Pittsburgh, PA, USA). Community profiles were subjected to cluster analysis using QuantityOne software (version 4.2; BioRad). Dice’s band-matching coefficient and unweighted pair group method with arithmetic averages (UPGMA) were employed to analyze the results.
3
Ubiquitin Probe Labeling in HEK293T Cells
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HEK293T cells were treated with a 5 μM
final concentration
of the indicated compounds for 24 h. Cells were lysed in HR lysis
buffer supplemented with protease inhibitor cocktail (11836145001,
Roche). Samples were kept on ice and lysed by sonication (10 cycles
of 30 s on and 30 s off). The protein extract (25 μg) was labeled
with either 1 μM Rh-Ub-PA probe or 0.5 μM Cy5-Ub-PA probe
for 30 min at 37 °C. For the cell lysate incubation, HEK293T
cells were lysated as described above. HEK293T cell lysates were preincubated
with a 5 μM final concentration of compounds for 1 h, followed
by incubation with a 0.5 μM Cy5-Ub-PA probe for 30 min at 37
°C. Labeling reactions were terminated with sample buffer and
heating to 100 °C for 10 min. Samples were size-separated in
SDS-PAGE gels. In-gel fluorescence signals were scanned by employing
the Typhoon FLA 9500 molecular imager (GE Healthcare). Images were
analyzed using ImageJ software.
final concentration
of the indicated compounds for 24 h. Cells were lysed in HR lysis
buffer supplemented with protease inhibitor cocktail (11836145001,
Roche). Samples were kept on ice and lysed by sonication (10 cycles
of 30 s on and 30 s off). The protein extract (25 μg) was labeled
with either 1 μM Rh-Ub-PA probe or 0.5 μM Cy5-Ub-PA probe
for 30 min at 37 °C. For the cell lysate incubation, HEK293T
cells were lysated as described above. HEK293T cell lysates were preincubated
with a 5 μM final concentration of compounds for 1 h, followed
by incubation with a 0.5 μM Cy5-Ub-PA probe for 30 min at 37
°C. Labeling reactions were terminated with sample buffer and
heating to 100 °C for 10 min. Samples were size-separated in
SDS-PAGE gels. In-gel fluorescence signals were scanned by employing
the Typhoon FLA 9500 molecular imager (GE Healthcare). Images were
analyzed using ImageJ software.
4
Histological Analysis of Murine Eye Tissue
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Upon completion of the imaging protocols, the animals were overdosed with inhaled isoflurane, decapitated, and the eyes removed for preservation. Each eye was fixed in 4% paraformaldehyde at least overnight and then transferred to a 70% ethanol solution. After removal of anterior chamber and lens, P14 eyes were imaged for green fluorescence on a Typhoon FLA 9500 molecular imager (GE, Piscataway, NJ, USA) and tumor area manually delineated and densitized using the Analysis Tools module of ImageQuant TL software. Whole P29 eyes were paraffin embedded and 5–7 µm sections were obtained using a microtome. Sections were then deparaffinized with xylene and rehydrated in ethanol from 100% to 70%. Sections were stained using a standard Mayer’s Hematoxylin and Eosin protocol (Sigma, St. Louis, MO, USA). Briefly, tissue sections were placed in 0.1% Mayer’s Hematoxylin (filtered prior to use) for 5 minutes and rinsed in cool running tap H2O for 5 minutes. Sections were then stained with Eosin (0.5% in 95% ethanol) for 2.5 minutes. Slides were dipped in tap H2O and then dehydrated in increasing concentrations of ethanol (70–100%) followed by drying in xylenes before mounting with Permount (Fisher Scientific, Pittsburgh, PA, USA). Images were collected using a DM2000 microscope with a DFC310 FX digital CCD color camera (Leica, Buffalo Grove, IL, USA).
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