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  • Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress

    For use with PURExpress® Δ (aa, tRNA) Kit (E6840), PURExpress® Δ Ribosome Kit (E3313), PURExpress® In Vitro Protein Synthesis Kit (E6800), and PURExpress® Δ RF123 Kit (E6850).

    Introduction

    Using TCA to precipitate labeled protein after synthesis in the presence of 35S methionine allows the measurement of radiolabel incorporation and provides a means to estimate the amount of protein synthesized in a reaction. When compared to a reaction without template DNA (negative control reaction), the overall efficiency of the protein synthesis reaction is revealed.

    Protocol

    1. Following incubation, mix 5 μl of the labeled PURExpress reaction with 250 μl of 1M NaOH in a glass test tube and incubate at RT for 10 min. NaOH will deacylate all charged tRNA’s, including 35S-Met-tRNA, to ensure that all TCA precipitable counts originate from labeled protein.

    2. Add 2 ml cold TCA/CAA mix (25% trichloroacetic acid/ 2% casamino acids) to sample and vortex briefly. Incubate on ice for 5 min. Acidifying the solution with TCA will precipitate all the protein.

    3. Use vacuum filtration to collect the precipitated protein. Pre-wet glass fiber filters with 10% TCA and transfer sample to the filter with vacuum. Rinse the tubes 3X with cold 10%TCA and transfer to the vacuum filter. Wash once with 95% ethanol to dry the filters and prevent quenching. 

      3a) Alternatively, soak 2.5 cm glass or paper filters in 10% TCA and allow to dry. Spot 20 μl of the base-treated reaction (step 1) on the filter and transfer to a beaker containing 100 ml ice-cold TCA and incubate w/ swirling for 15 minutes on ice. Repeat wash three times (total), then wash with ethanol and dry.

    4. Place dry filters into scintillation vials with 2 ml scintillation fluid.

    5. Prepare a control filter to measure the total counts in a labeling reaction. Directly pipet 5 μl of a reaction onto a dry glass fiber filter and place the filter into scintillation fluid.

    6. Measure samples in a scintillation counter. Multiply all values by 5 to determine the counts in a 25 μl reaction. The TCA precipitated counts is a measure of the efficiency of the labeling and can be represented as a percentage of the total counts by dividing the TCA sample value by the total counts control filter value and multiplying by 100.

      35S is not a strong isotope. The signal may be quenched by extra salt, H2O, etc. in the sample. We recommend recounting the samples after the filters have been soaked in the scintillation fluid for 4 hours to overnight. We find this second measurement is often more consistent and reliable.