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  • Determination of Protein Synthesis Yield with PURExpress (E3313)

    Introduction

    Using the equations below, one can calculate the yield of protein synthesized in the reaction. The calculations do not differentiate full-length protein from truncated products and as such, all translation products contribute to the calculation of yield. Prior to using the equations, it is necessary to have determined the number of picomoles of both labeled and unlabeled methionine in the reaction, the number of counts produced by no template (background), target protein (TCA-precipitable) and the total counts in a reaction.

    picomoles of Met: 

    unlabeled = 0.3 mM in reaction = 300 pmol/μl x 25 μl rxn = 7,500 pmol 

    labeled = 2 μl of 35S-Met (15 mCi/ml, 1,000 Ci/mmol) per rxn = 2 μl x 15 mCi/ml = 30 μCi x μmol/1 x 106 μCi = 3 x 10-5 μmol = 30 pmol 

    total = 7,500 pmol unlabeled Met + 30 pmol labeled Met = 7,530 pmol Met 

    Total counts = total cpm per 5 μl control x (reaction volume/5) 

    Specific Activity = Total counts / pmoles methionine (labeled and unlabeled) 

    Met incorporation (pmoles) = [(TCA ppt cpm-background cpm) x total reaction volume/5] / Specific Activity 

    pmoles of protein = pmoles of incorporated Met / # of Met residues in target protein 

    Yield of protein (μg) = pmoles of protein x MW of protein / 106

    Example of Calculation: 

    DHFR: 
    17,998 Daltons, 5 methionine residues 

    CPM's Measured: 
    1.12 x 107 total, 5 μl aliquot
    1.8 x 104 background
    1.95 x 106 TCA ppt 

    Total Counts = 1.12 x 107 cpm x 5 = 5.6 x 107 cpm/25 μl rxn 

    Specific Activity = 5.6 x 107cpm / 7530 pmol = 7437 cpm/pmol

    Methionine Incorporation = (1.95 x 106 cpm – 1.8 x 104 cpm) x 5 / 7437 = 1297 pmol Met 

    pmoles DHFR = 1297 pmol Met / 5 Met/DHFR 

    Yield (μg) = 259.4 x 17,998 / 106 = 4.67 μg/25 μl rxn x 40/ml = 187 μg/ml