The day before the experiment, HEK293T cells stably expressing MyrPB-ezrin-rLucII and rGFP-CAAX were suspended in DMEM/F12 without phenol red (#21041025; Invitrogen) supplemented with 1% FBS, sifted through a 70-µm cell strainer (#352350; Falcon), and seeded in white 384-well culture plates (#3570; Corning). After an overnight incubation at 37°C with 5% CO2, compounds from different libraries (Microsource Discovery, Biomol GmbH, Prestwick, and Sigma-Aldrich) were added using an Echo 555 acoustic dispenser (Labcyte) at a final concentration of 10 µM for 1 h. Substrate solution (coelenterazine 400a diluted in HBSS with 1% pluronic acid) was added 15 min before reading at a final concentration of 2.5 µM. BRET signals were monitored with a Synergy NEO HTS microplate reader (BioTek) equipped with a donor filter of 410/80 nm and an acceptor filter of 515/40 nm.
Synergy neo hts microplate reader
Manufactured by Agilent Technologies
The Synergy NEO HTS microplate reader is a high-throughput, multi-mode plate reader designed for a variety of applications in life science research. It is capable of performing absorbance, fluorescence, and luminescence measurements on 6- to 384-well microplates.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using synergy neo hts microplate reader
1
Measuring Membrane Protein Interactions
2
Enzymatic Assay for ACAT1 Activity
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Recombinant
ACAT1 (200 ng/mL in
PBS) or vehicle was incubated with 2 mM MGO or vehicle for 2 h at
37 °C. 10 mM aqueous stocks of coenzyme A trilithium salt (CoA,
Sigma Aldrich) and acetoacetyl coenzyme A (AcAc-CoA, Cayman Chemicals)
were added to 40 mM MgCl2 in PBS to give 400 μM final
concentration of each substrate. The substrate mixture was allowed
to preequilibrate at 37 °C, then mixed in a 1:1 ratio with the
reaction solutions, and imaged at 303 nm33 (link) once a minute using a Synergy Neo HTS Microplate Reader (BioTek).
Relative AcAc-CoA consumption for each condition was calculated by
the normalized absolute difference between the 303 nm absorbance in
the ACAT1 containing reactions and the corresponding ACAT1-less control
reactions.
For MS/MS monitoring of Ac-CoA production, aliquots
of the reactions were taken at the indicated time points and mixed
with MeOH in a 4:1 MeOH/sample ratio to quench the enzymatic reaction.
Internal deuterated standards, 1 μL of 10 mM d3-serine, were
added to the extraction solution for sample normalization, the samples
were centrifuged for 15 min at 16,000g, and the supernatant
was kept for immediate LC–MS/MS analysis.
ACAT1 (200 ng/mL in
PBS) or vehicle was incubated with 2 mM MGO or vehicle for 2 h at
37 °C. 10 mM aqueous stocks of coenzyme A trilithium salt (CoA,
Sigma Aldrich) and acetoacetyl coenzyme A (AcAc-CoA, Cayman Chemicals)
were added to 40 mM MgCl2 in PBS to give 400 μM final
concentration of each substrate. The substrate mixture was allowed
to preequilibrate at 37 °C, then mixed in a 1:1 ratio with the
reaction solutions, and imaged at 303 nm33 (link) once a minute using a Synergy Neo HTS Microplate Reader (BioTek).
Relative AcAc-CoA consumption for each condition was calculated by
the normalized absolute difference between the 303 nm absorbance in
the ACAT1 containing reactions and the corresponding ACAT1-less control
reactions.
For MS/MS monitoring of Ac-CoA production, aliquots
of the reactions were taken at the indicated time points and mixed
with MeOH in a 4:1 MeOH/sample ratio to quench the enzymatic reaction.
Internal deuterated standards, 1 μL of 10 mM d3-serine, were
added to the extraction solution for sample normalization, the samples
were centrifuged for 15 min at 16,000g, and the supernatant
was kept for immediate LC–MS/MS analysis.
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