We measured BCKD activity using a modified version of a previous assay (White et al., 2018 (link)). Approximately 30 mg of frozen gastrocnemius muscle or liver were crushed in liquid nitrogen and homogenized (Bio‐Gen PRO200 Homogenizer, PRO Scientific, Oxford, CT) in 250 μL of ice‐cold buffer 1 (30 mM potassium phosphate buffer (KPI), 3 mM EDTA, 5 mM DTT, 1 mM valine, 3% FBS, 5% Triton X‐100 and 1 μm leupeptin (Sigma Aldrich, #L2884)). The resulting sample was centrifuged (10 min at 10,000 g, 4°C) and 50 μL of the supernatant was added to 300 μL of buffer 2 (50 mM HEPES, 30 mM KPI, 0.4 mM CoA (Sigma Aldrich, #C4282), 3 mM NAD+ (Sigma Aldrich, #N0632), 5% FBS, 2 mM Thiamine (Sigma Aldrich, #T1270), 2 mM magnesium chloride and 7.8 μM [14C] valine (Perkin Elmer, #NEC291EU050UC, Waltham, MA)). This reaction took place in a 1.5‐mL Eppendorf tube containing a raised 2‐M NaOH wick trap. Each Eppendorf tube was sealed tight and placed in a shaking incubator at 37°C for 30 min. The radiolabeled 14CO2 contained in the wick trap was counted in a liquid scintillation counter.
Nec291eu050uc
Manufactured by PerkinElmer
The NEC291EU050UC is a laboratory instrument manufactured by PerkinElmer. It is designed to perform various analytical tasks in a research or testing environment. The core function of this product is to provide precise measurements and data analysis capabilities to support scientific investigations and experiments. No further details or interpretation about the intended use of this product can be provided in this unbiased and factual description.
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4 protocols using nec291eu050uc
1
Measuring BCKD Activity in Muscle and Liver
2
Quantifying Long-Lived Protein Degradation
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The degradation of long-lived proteins in fed RPE-1 cells was done according to a protocol described previously (Engedal et al., 2013 (link)). In brief, cells seeded in six-well plates were transfected with siRNA duplexes on the same day and radiolabeled with 0.25 µCi/ml L-[14C] valine (NEC291EU050UC; PerkinElmer) in RPMI 1640 (R2405; Sigma-Aldrich) and 10% FBS for 24 h. Cells were then washed and chased with 1.5 ml complete RPMI 1640 supplemented with 10 mM cold valine (V0500; Sigma-Aldrich) for 3 h to allow degradation of short-lived proteins. Then the cells were incubated with 1 ml complete DMEM (10% FBS; supplemented with 10 mM cold valine) for 20 h to allow degradation of long-lived proteins. Subsequently, proteins were precipitated by 50% TCA (100807; Merck Millipore). Supernatant (degraded proteins, TCA-soluble fraction) and pellet (nondegraded protein, TCA-nonsoluble fraction) were obtained through centrifugation at 5,000 g for 10 min at 4°C. The pellet was solubilized in 500 µl 0.2 M KOH (105033; Merck Millipore). Radioactivity was determined by liquid scintillation counting. The degradation rate for long-lived proteins was calculated as the percentage of radioactivity in the TCA-soluble fraction relative to the total radioactivity in the TCA-soluble and nonsoluble fractions, divided by the incubation time.
3
Long-Lived Protein Degradation Assay
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Cells were seeded in 6-well plates and cultured for 24 h. After treatments, cells were maintained with 0.2 lCi/mL [14C]-valine (NEC291EU050UC, PerkinElmer) for 18 h at 37°C. After several washing steps in PBS (pH 7.4), the medium with the isotope-labeled amino acid was changed for a cell culture medium with l -valine (V0153, Sigma-Aldrich), first for 1h (prechase), and then in combination with several experimental treatments (chase) for 4 h. Thereafter, proteins were precipitated with 10% (v/v) trichloroacetic acid (TCA) (T9159, Sigma-Aldrich), separated from the soluble radioactive fraction by centrifugation at 6000 g for 20 min and then dissolved in 0.2 N NaOH. Finally, the long-lived protein degradation was measured by adding a small quantity of each sample into a scintillation liquid and measuring the radioactive activity with a liquid scintillation counter.
4
Measurement of Long-Lived Protein Degradation
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Long-lived protein degradation assay was carried out as described previously [43 (link)], Briefly, MEFs were cultured in 24-well plates overnight, L-[14C]-valine (PerkinElmer, NEC291EU050UC) was added to a final concentration of 0.2 µCi/ml to label intracellular proteins. Cells were incubated for 18–24 h before changing to fresh medium for another hour with 10% cold l -valine to deplete labeled short-lived proteins. The cells were then incubated in EBSS or DMEM (plus 0.1% of BSA and 10 mM of valine) with or without J3 for an additional 6 or 16 h. The culture medium was recovered, from which the degraded long-lived proteins were measured via liquid scintillation.
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