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Synergy mx monochromator

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The Synergy Mx Monochromator is a lab equipment product from Agilent Technologies. It is a device used to separate light into its component wavelengths.

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Lab products found in correlation

Celltiter 96 aqueous one solution reagent, by Promega (1 mentions) Annexin 5 fitc apoptosis kit, by Abcam (1 mentions) Annexin 5 fitc assay kit, by Cayman Chemical (1 mentions) Mtt reagent, by Merck Group (1 mentions)

Spelling variants of this product

Synergy Mx (710 citations) Synergy Mx Monochromator-Based Multi-Mode Microplate Reader (23 citations) Synergy Mx Monochromator-based Microplate Reader (4 citations) Synergy Mx Monochromator-Based (2 citations)

8 protocols using synergy mx monochromator

1

Hsp90-Mediated Refolding of Luciferase

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Firefly luciferase was heat denatured at 45 °C fo r 8 min in 20 mM Hepes, pH 7.6, 85 mM potassium acetate, 1 mM magnesium acetate, 1.5 mM DDT and 1 mg/ml BSA containing 10 μM human Hsp90β. The refolding of thermally inactivated luciferase was initiated by diluting it 10-fold in tubes containing an ATP regenerating system (creatine phosphokinase (3.5U/ml), phosphocreatine (10 mM) and ATP (2 mM)) and Hsp70 (4 μM), Hsp40 (2 μM), Bag1 (1.5 μM), HOP (0.5 μM), and Aha1 (2 μM), in the presence or absence of SEW84 (15 μM), GA (15 μM) or JG98 (15 μM). The reaction was carried out for 8 h at RT. Luciferase activity was measured using Promega Steady-Glo® luciferase assay system and BioTek Synergy Mx Monochromator based multi-mode plate reader. Native luciferase was set as 100% and all other values normalized to this control.
Singh J.K., Hutt D.M., Tait B., Guy N.C., Sivils J.C., Ortiz N.R., Payan A.N., Komaragiri S.K., Owens J.J., Culbertson D., Blair L.J., Dickey C., Kuo S.Y., Finley D., Dyson H.J., Cox M.B., Chaudhary J., Gestwicki J.E, & Balch W.E. (2020). Management of Hsp90-Dependent Protein Folding by Small Molecules Targeting the Aha1 Co-Chaperone. Cell chemical biology, 27(3), 292-305.e6.
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2

Aha1-mediated Hsp90 ATPase Kinetics

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The indicated chaperones (Hsp90 (8 μM), Hsp70 (4 μM), Hsp40 (2 μM), BAG1 (1.5 μM), HOP (0.5 μM) and Aha1 (2 μM)) were added to the wells of a 384-well plate containing reaction buffer (25 mM Hepes pH 7.5, 10 mM MgCl2, and 1 mM DTT) and incubated on ice for 30 min. Following the incubation period, vehicle or 15 μM SEW84 were added to the reaction mixture and the reaction allowed to continue for an additional 30 min on ice. Subsequently, 2 mM ATP was added to the reaction mixture and the plate immediately transferred to 37 °C to initiate ATPase activity. To assess the rate of ATP hydrolysis, 7 μL aliquots were withdrawn at 0, 3, 6, 15, 30, and 60 min and transferred to the wells of a white 384 well microtiter plate containing 3 μL of EDTA (100 mM) on ice. After completion of the time course, 15 μL of the QR reagent was added to each well followed immediately by 2.5 μL of 35% sodium citrate and mixed. Kinetics of Aha1 stimulated Hsp90 ATP hydrolysis was measured using a BioTek Synergy Mx Monochromator based multi-mode plate reader.
Singh J.K., Hutt D.M., Tait B., Guy N.C., Sivils J.C., Ortiz N.R., Payan A.N., Komaragiri S.K., Owens J.J., Culbertson D., Blair L.J., Dickey C., Kuo S.Y., Finley D., Dyson H.J., Cox M.B., Chaudhary J., Gestwicki J.E, & Balch W.E. (2020). Management of Hsp90-Dependent Protein Folding by Small Molecules Targeting the Aha1 Co-Chaperone. Cell chemical biology, 27(3), 292-305.e6.
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3

Aha1-Mediated Regulation of Hsp90 ATPase

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Hsp90 (8 μM) was added to the wells of a 384-well plate containing reaction buffer (25 mM Hepes pH 7.5, 10 mM MgCl2, and 1 mM DTT) and incubated on ice for 30 min in the absence and presence of Aha1 (16 μM). Following the incubation period, different concentrations of SEW84 (0, 0.1, 0.5, 1, and 5 μM), or its derivatives, were added to the reaction mixture and the reaction allowed to continue for an additional 30 min on ice. Subsequently, 2 mM ATP was added to the reaction mixture and the plate immediately transferred to 37 °C to initiate ATPase activity. To assess the rate of ATP hydrolysis, 7 μL aliquots were withdrawn at 0, 3, 6, 15, 30, and 60 min and transferred to the wells of a white 384 well microtiter plate containing 3 μL of EDTA (100 mM) on ice. After completion of the time course, 15 μL of the QR reagent was added to each well followed immediately by 2.5 μL of 35% sodium citrate and mixed. Kinetics of Aha1 stimulated Hsp90 ATP hydrolysis was measured using a BioTek Synergy Mx Monochromator based multi-mode plate reader.
Singh J.K., Hutt D.M., Tait B., Guy N.C., Sivils J.C., Ortiz N.R., Payan A.N., Komaragiri S.K., Owens J.J., Culbertson D., Blair L.J., Dickey C., Kuo S.Y., Finley D., Dyson H.J., Cox M.B., Chaudhary J., Gestwicki J.E, & Balch W.E. (2020). Management of Hsp90-Dependent Protein Folding by Small Molecules Targeting the Aha1 Co-Chaperone. Cell chemical biology, 27(3), 292-305.e6.
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4

Cell Viability and Apoptosis Assay

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Cells were seeded at a density of 5000 cells/well then treated at the doses indicated. 72 hours later, cells were incubated with CellTiter 96 Aqueous One Solution Reagent (Promega), and viability was assessed using a Synergy Mx Monochromator (BioTek Instrument Inc.). Flow cytometry-based apoptosis assays were performed with the Annexin V-FITC Apoptosis Kit (Biovision), following the manufacturer’s instructions. Multiwell plate apoptosis assays were performed with the Annexin V FITC Assay Kit (Cayman Chemical).
Cheng Y., Holloway M.P., Nguyen K., McCauley D., Landesman Y., Kauffman M.G., Shacham S, & Altura R.A. (2014). XPO1 (CRM1) inhibition represses STAT3 activation to drive a survivin-dependent oncogenic switch in triple negative breast cancer. Molecular cancer therapeutics, 13(3), 675-686.
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5

Mitochondrial Activity and Cell Death

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To estimate cell death due to loss of mitochondrial activity at the end of PAMP treatments, the cells were incubated with 100 μl MTT reagent (Sigma Chemical Co.) at 37 °C for 3 h. The metabolic activity of viable cells was measured at 570 nm using a BioTek microplate reader (Synergy Mx Monochromator, Winooski, VT, USA).
Chang S.C, & Ding J.L. (2014). Ubiquitination by SAG regulates macrophage survival/death and immune response during infection. Cell Death and Differentiation, 21(9), 1388-1398.
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6

ATP Measurement in Bacterial Cultures

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ATP measurements were performed using the BacTiter-Glo microbial cell viability assay according to the manufacturer’s instructions. Briefly, cells were washed to remove any potential ATP contamination present in the growth medium. Equal volumes of the cell suspension and the BacTiter-Glo reagent were mixed and incubated for 5 min before measuring luminescence using a Synergy Mx monochromator-based multimode microplate reader (BioTek). Signal intensities were corrected for cell numbers by dividing the luminescence signal by the OD at 595 nm of the cell suspension. ATP levels were modulated by adding different concentrations of arsenate (0.1, 1, and 10 mM) to bacterial cultures and measuring at different time points, and hence in different growth phases of the culture.
Dewachter L., Bollen C., Wilmaerts D., Louwagie E., Herpels P., Matthay P., Khodaparast L., Khodaparast L., Rousseau F., Schymkowitz J, & Michiels J. (2021). The Dynamic Transition of Persistence toward the Viable but Nonculturable State during Stationary Phase Is Driven by Protein Aggregation. mBio, 12(4), e00703-21.
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7

Quantifying Hsp90β-Aha1 Interaction Kinetics

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Acrylodan-labeled full length or CTD-Aha1 (1 μM) was incubated with the 25 μM SEW84 in the presence or absence of different amounts of Hsp90 (0 to 10 μM) for 30 min over ice in reaction buffer (20 mM Hepes, pH 7.5, 10 MgCl2, 5 mM KCl). The excitation and emission spectra were recorded at 390 nm, and 495 nm, respectively; using BioTek Synergy Mx Monochromator based multi-mode plate reader. The increased acrylodan-Aha1 signals at 495 nm upon binding to Hsp90β was used to determine the dissociation constant (Kd) of the Aha1 and Hsp90β interaction using the equation:
ΔF=ΔFmaxL/(Kd+L)
Where ΔF is change in the fluorescence intensity of the acrylodan-Aha1 upon binding to Hsp90β. ΔFmax is the maximum change in the fluorescence intensity of acrylodan-Aha1 when it is fully liganded with Hsp90β, and L is the concentration of Hsp90β. The ΔFmax value (50091 ± 4201) was calculated. ΔF was calculated by subtracting the fluorescence intensity of acrylodan-Aha1 in the absence of Hsp90β from the fluorescence intensity of acrylodan-Aha1 in the presence of Hsp90β (Singh et al., 2007 (link)). All the data were statistically analyzed by One-Way ANOVA and curve fitted using GraphPad software.
Singh J.K., Hutt D.M., Tait B., Guy N.C., Sivils J.C., Ortiz N.R., Payan A.N., Komaragiri S.K., Owens J.J., Culbertson D., Blair L.J., Dickey C., Kuo S.Y., Finley D., Dyson H.J., Cox M.B., Chaudhary J., Gestwicki J.E, & Balch W.E. (2020). Management of Hsp90-Dependent Protein Folding by Small Molecules Targeting the Aha1 Co-Chaperone. Cell chemical biology, 27(3), 292-305.e6.
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8

Hydrolytic Activity of CTX-M-14 and KPC-2 Enzymes

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CTX-M-14 and KPC-2 were purified as previously described35 ,36 (link),44 (link). The hydrolytic activity of CTX-M-14 and KPC-2 was determined using the β-lactamase substrate nitrocefin in a PBS-P+ buffer containing 200 mM phosphate buffer pH 7.4, 27 mM potassium chloride and 1.37 M sodium chloride, 0.5% (v/v) surfactant P20 supplemented with 2% DMSO. Nitrocefin hydrolysis was monitored using a BioTek Synergy Mx monochromator-based multimode microplate reader at 486 nm wavelength. For CTX-M-14 and KPC-2 inhibition assays, the nitrocefin concentration used was 20 µM and 10 µM, respectively. The Km of nitrocefin for CTX-M-14 is 22 µM and for KPC-2 is 10 µM. Compounds were used for IC50 measurements up to 3 mM based on their solubility in DMSO. The final protein concentration used in the reaction for CTX-M-14 and KPC-2 was 0.3 nM and 1 nM respectively. The protein was added last to initiate the reaction.
Kroeck K.G., Sacco M.D., Smith E.W., Zhang X., Shoun D., Akhtar A., Darch S.E., Cohen F., Andrews L.D., Knox J.E, & Chen Y. (2019). Discovery of dual-activity small-molecule ligands of Pseudomonas aeruginosa LpxA and LpxD using SPR and X-ray crystallography. Scientific Reports, 9, 15450.
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