Samples were prepared in NuPage™ LDS loading buffer and reducing reagent, boiled for 5 min and electrophoresed on 12% NuPage Bis-Tris gels (all Invitrogen Life Technologies). Gels were stained with Lumitein stain (Biotium, CA, USA) or silver stain (Sigma) according to manufacturer's instructions and visualised using a typhoon fluorescent scanner (GE Healthcare), or protein was transferred to nitrocellulose membranes and blocked using PBS with 5% milk (w/v) and 0.2% Tween20 (v/v). Hybridisation was overnight at 4 °C with anti-ps20 primary antibodies at 1:500 dilution and secondary anti-rabbit at 1:2000 dilution, simultaneously. Washing was with 10 ml PBS with 0.2% Tween20 (v/v) at room temperature 6 times for 10 min followed by incubation with ECL substrate (Thermo scientific) and acquisition using the Imagequant™ system (GE Healthcare).
Typhoon fluorescent scanner
Manufactured by GE Healthcare
Sourced in United States
The Typhoon fluorescent scanner is a device designed for the detection and analysis of fluorescent signals in various biological samples. It utilizes a laser-based detection system to capture high-resolution images of fluorescently labeled molecules, such as proteins, nucleic acids, or other biomolecules. The Typhoon scanner is capable of providing accurate and sensitive quantitative data, making it a valuable tool for researchers in fields like molecular biology, biochemistry, and proteomics.
Automatically generated - may contain errors
Lab products found in correlation
His6 e1, by R&D Systems (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 maleimide, by Thermo Fisher Scientific (1 mentions)
Clarity lightning ecl, by Bio-Rad (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Mouse anti ubiquitin, by Fortrea (1 mentions)
5 protocols using typhoon fluorescent scanner
1
Protein Analysis via SDS-PAGE and Western Blot
2
Characterizing Parkin Ubiquitination Activity
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UbcH7 (10 μg) was charged with N-terminal fluorescein-labelled Ub (10 μg FluoUb) using 0.02 μg His6-E1 (Boston Biochem Inc.), 0.5 mM ATP and 10 mM MgCl2 in a 100 μl reaction (in 50 mM Tris/HCl pH 7.4, 120 mM NaCl and 1 mM TCEP). After 60 min incubation at 30 °C, 0.8 μg of UbcH7∼FluoUb was mixed with 1.2 μg of WT or mutant RnParkin (in 50 mM Tris/HCl pH 7.4, 120 mM NaCl and 1 mM TCEP). Mixtures were incubated for 20 min at 30 °C for discharging. Reactions were stopped with the addition of SDS–PAGE sample buffer containing 12 mM TCEP, resolved on SDS–PAGE and analysed using the Typhoon fluorescent scanner (GE) and Coomassie staining. Fluorescence strength of UbcH7∼FluoUb was measured for quantification using ImageJ.
3
Fluorescent Labeling of Nanobodies
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As for PCMB labelling, the Nbs were transferred into a nonreducing buffer (10 mM HEPES pH 7.6, 50 mM NaCl) using a PD-10 desalting column. Nbs were mixed with a 1.5-fold molar excess of Alexa Fluor 488 maleimide (Thermo Fisher) and incubated on ice for 1 h before quenching with a 100-fold molar excess of DTT. The samples were analysed by nonreducing SDS–PAGE and fluorescence detection of the resulting acrylamide gel on a Typhoon fluorescent scanner (GE Healthcare).
4
Ubiquitin Activation and Parkin Discharge Assay
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Ten micrograms of ubiquitin-conjugating enzyme, UbcH7, were charged with 10 µg ubiquitin or N-terminally fluorescein-labelled Ub (FluoUb, Boston Biochem) using 0.02 µg ubiquitin-activating enzyme (His6-E1, Boston Biochem Inc.), 0.5 mM ATP and 10 mM MgCl2 in 50 mM Tris-HCl pH 7.4, 120 mM NaCl and 1 mM TCEP, in a 100 µl reaction. Reactions were performed at 37°C at different time points to a maximum of 60 min.
Ubiquitin-loaded UbcH7 (0.8 µg UbcH7–Ub or UbcH7–FluoUb) was mixed with 1.2 µg of RnParkin (in 50 mM Tris-HCl pH 7.4, 120 mM NaCl and 1 mM TCEP). Mixtures were incubated at 30°C for up to 40 min to monitor discharging (figure 2 a,c). Reactions were stopped with the addition of SDS-PAGE sample buffer containing 12 mM TCEP, resolved on SDS-PAGE and analysed using the Typhoon fluorescent scanner (GE), Coomassie staining, silver staining (Thermo Pierce kit) or transferred to a nitrocellulose membrane and probed with mouse anti-ubiquitin (Covance, 1 : 10 000) and detected with Clarity Lightning ECL (Bio Rad). Images acquired with an ImageQuant LAS 500 (GE Healthcare). Densitometry and fluorescence intensity analysis was performed using Fiji [83 (link)].
Ubiquitin-loaded UbcH7 (0.8 µg UbcH7–Ub or UbcH7–FluoUb) was mixed with 1.2 µg of RnParkin (in 50 mM Tris-HCl pH 7.4, 120 mM NaCl and 1 mM TCEP). Mixtures were incubated at 30°C for up to 40 min to monitor discharging (
5
Comprehensive Lipid Profiling of M. bovis
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The M. bovis strains were grown to an OD600 of 0.5 in 7H9 medium with ADP at pH 7 followed by addition 1% of 14 C acetate and incubation for 24 h under agitation at 37ºC. Then, the bacteria were pelleted, washed in PBS and the lipids were extracted once overnight in 1:2 (chloroform: methanol) followed by two overnight extractions in 2:1 (chloroform: methanol). Upon Folch wash, 14C incorporation was determined in the total extractable lipids by scintillation counting. Thin layer chromatography (TLC) plates were used to analyse lipid samples, by spotting 10000-15000 counts per minute (CPM) of each lipid sample. The petroleum ether: acetone system (98:2) was used to resolve PDIMs [8] (link). Labelled lipids were detected using Typhoon fluorescent scanner (GE Healthcare). Lipid extractions and TLCs were repeated in four independent biological replicates with similar findings. Unlabelled lipids were obtained following the same extraction protocol described above from cultures grown until stationary phase at neutral pH. Unlabelled lipids were analysed by LC/MS as previously described [11] (link).
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