M1000 spectrophotometer

Manufactured by Tecan

The M1000 spectrophotometer is a versatile instrument designed for accurate absorbance measurements. It features a wavelength range of 230 to 1000 nm and can perform a variety of spectroscopic analyses. The M1000 provides reliable and precise data for a wide range of applications in life science research and clinical diagnostics.

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Lab products found in correlation

Enzchek pyrophosphate assay kit, by Thermo Fisher Scientific (2 mentions) Chloramphenicol, by Merck Group (1 mentions) Kanamycin, by Merck Group (1 mentions) Fortessa flow cytometer, by BD (1 mentions) Ampicillin, by Merck Group (1 mentions)

5 protocols using m1000 spectrophotometer

BCL-2 BH4 SAHBA Inhibits BAX Activation

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Example 4

BCL-2 BH4 SAHB_Aand unmodified BCL-2 BH4 peptide were incubated with 500 nM recombinant BAX protein and 5 μL of liposomes containing entrapped fluorophore (8, aminonaphthalene-1,3,6-trisulfonate (ANTS)) and quencher (p-xylene-bis-pyridinium bromide (DPX)), generated as previously described in Cohen et al. (Chem. Biol. 19:1175-1186, 2012). The reaction mixture was heated to 37° C. while fluorescence was measured at the indicated time points using a Tecan M1000 spectrophotometer (excitation wavelength of 355 nm and an emission wavelength of 520 nm). The BCL-2 BH4 SAHB_Adose-responsively decreased heat-induced activation of BAX and BAX-mediated poration, whereas the unmodified BH4 peptide had no such effect (FIG. 12).

Additional experiments were performed to determine whether BCL-2 BH4 SAHB_Awould also inhibit BIM SAHB_A-mediated activation of BAX protein. In these experiments, the BCL-2 BH4 SAHB_Aand unmodified BCL-2 BH4 peptide were incubated with 1.5 μM BIM SAHB_A(Gavathiotis et al., Nature 455:1076-1081, 2008), 0.75 μM recombinant BAX protein, and 5 μL liposomes loaded with the ANTS and DPX (described above) at a variety of different concentrations. The data show that BCL-2 BH4 SAHB_Adose-responsively decreased BAX activation in response to BIM SAHB_Atreatment, whereas the unmodified BH4 peptide exhibited a modest inhibitory effect (FIG. 13).

US11046739B2. BH4 stabilized peptides and uses thereof (2021-06-29). Dana-Farber Cancer Institute, Inc. [US]. Inventors: Loren D. Walensky [US], Michelle L. Stewart [US], Lauren Barclay [US].

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Modulation of BAX Activation by BCL-2 BH4 SAHB

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Example 4

BCL-2 BH4 SAHB_Aand unmodified BCL-2 BH4 peptide were incubated with 500 nM recombinant BAX protein and 5 μL of liposomes containing entrapped fluorophore (8, aminonaphthalene-1,3,6-trisulfonate (ANTS)) and quencher (p-xylene-bis-pyridinium bromide (DPX)), generated as previously described in Cohen et al. (Chem. Biol. 19:1175-1186, 2012). The reaction mixture was heated to 37° C. while fluorescence was measured at the indicated time points using a Tecan M1000 spectrophotometer (excitation wavelength of 355 nm and an emission wavelength of 520 nm). The BCL-2 BH4 SAHB_Adose-responsively decreased heat-induced activation of BAX and BAX-mediated portion, whereas the unmodified BH4 peptide had no such effect (FIG. 12).

US10106590B2. BH4 stabilized peptides and uses thereof (2018-10-23). Dana-Farber Cancer Institutes, Inc. [US]. Inventors: Loren D. Walensky [US], Michelle L. Stewart [US], Lauren Barclay [US].

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Measuring Bacterial YFP Expression

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Transformed strains were grown overnight at 37°C and 200 RPM in LB Miller supplemented with 10 μg/ml chloramphenicol, kanamycin, and ampicillin (Sigma-Aldrich). 5 μl of cultures were diluted into fresh selective media in a 96-well microplate, incubated, and shaken at 37°C in a TECAN M1000 spectrophotometer. Serial dilutions were performed twice to maintain cells in the exponential phase of growth for a 12 hour period. 10 μl samples were extracted after the second and third serial dilutions and added to 200 μl Phosphate buffered saline (PBS) supplemented with 2 mg/mL kanamycin for halting growth. Single-cell YFP fluorescence from at least 20,000 cells were recorded by an BD Fortessa flow cytometer. The average YFP expression level was determined by taking the average of the fluorescence distribution and subtracting the average auto-fluorescence of E. coli pir116.

Farasat I, & Salis H.M. (2016). A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation. PLoS Computational Biology, 12(1), e1004724.

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Prenyltransferase Activity Assay for CymD

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The prenyltransferase activity of CymD was measured using the EnzChek Pyrophosphate Assay Kit (Invitrogen). The reaction buffer was composed of 50 mM Tris-HCl (pH 7.5), 1 mM MgCl₂, and 0.1 mM sodium azide. The 100-μL reaction mixture contained (final concentrations): 200 μM 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), 0.1 unit of purine nucleoside phosphorylase (PNPase), 2 units of inorganic pyrophosphatase (IPPase), 1 μM CymD, and 80 μM DMAPP. The assay was initiated by adding prenyl acceptor substrate to a final concentration of 500 μM. The reaction was monitored in triplicate in a flat-bottom transparent 96-well plate using a Tecan M1000 spectrophotometer. Reaction velocity was calculated from the increase in A₃₆₀ within the linear range.

Roose B.W, & Christianson D.W. (2019). Structural Basis of Tryptophan Reverse N-Prenylation Catalyzed by CymD. Biochemistry, 58(30), 3232-3242.

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Enzymatic Pyrophosphate Assay for DMATS1

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The EnzChek Pyrophosphate Assay Kit (Invitrogen) was used to evaluate the prenyltransferase activity of wild-type and mutant DMATS1 enzymes. 100 μL reactions were performed in assay buffer [50 mM Tris HCl (pH 7.5), 1.0 mM MgCl₂, 0.1 mM NaN₃]. Added to each reaction was 200 μM 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), 1 U purine nucleoside phosphorylase, 0.03 U inorganic pyrophosphatase, 0.1 μM DMATS1, and 80 μM prenyl donor (DMAPP or GPP). The assay was initiated upon the addition of prenyl acceptor substrate to a final concentration of 500 μM. Reactions containing no prenyl acceptor were included as negative control. Assays were performed in triplicate in a 96-well clear, flat-bottom plate, and monitored at A₃₆₀ on a Tecan M1000 spectrophotometer. Initial velocity was calculated using the ΔA₃₆₀ within the linear range.

Eaton S.A., Ronnebaum T.A., Roose B.W, & Christianson D.W. (2022). Structural Basis of Substrate Promiscuity and Catalysis by the Reverse Prenyltransferase N-dimethylallyl-l-Tryptophan Synthase from Fusarium fujikuroi. Biochemistry, 61(18), 2025-2035.

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