Exp_2_Key - MCB120L Experiment 2 Write-up KEY Overlay...

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MCB120L Winter 2009 Morand Experiment 2 Write-up KEY Overlay Spectra 1. Calculate the molar extinction coefficients, ε , of tyrosine and tryptophan at 280 nm. (Abs 280 / 0.25 mM x 1 cm)(10 -3 M / mM) = _____ Trp (W) ~ 5600 M -1 cm -1 Tyr (Y) ~ 1340 M -1 cm -1 2. Calculate the absorption coefficients, a , in units of (mg/ml) -1 cm -l of BSA, lysozyme and gelatin at 280 nm. lysozyme (1.0 / 0.4 mg/ml x 1 cm) = 2.5 (mg/ml) -1 cm -l BSA (0.53 / 0.8 mg/ml x 1 cm) = 0.66 (mg/ml) -1 cm -l gelatin (0.28 / 2.0 mg/ml x 1cm) = 0.14 (mg/ml) -1 cm -l 3. Briefly explain why 595 nm is used for the Bradford assay. Examination of the overlay spectra shows the greatest Abs (± protein) occurs at 595 nm, thus 595 nm is the most sensitive λ available for measuring the Abs. Bradford and A280 Protein Assays 1. Attach the standard curves for both assays. A computer plotting program may be used to plot the data to obtain the equation for the straight line or the curve (a second-order polynomial, for the Bradford plot, if needed). These equations usually are helpful in determining the quantity or concentration of protein in your unknown samples. A280:
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Exp_2_Key - MCB120L Experiment 2 Write-up KEY Overlay...

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