23 Jul CALCULATE THE AMOUNT OF PROTEIN ADDED IN EACH CASE FOR THE 500 UG/ML STANDARD SAMPLES.
One quantitative method for the measurement of protein uses a reagent called Coomassie Brilliant Blu Show more One quantitative method for the measurement of protein uses a reagent called Coomassie Brilliant Blue which changes from a brown to blue color upon binding protein. The blue color can be measured using a spectrophotometer (see lab 6). A cell biology researcher wishes to know the concentration of protein in an unknown sample and performs the following experiment: Protein Sample Protein Sample Protein Sample Volume protein added Volume water added Absorbance @595 nm 500 ug/ml protein standard 500 ug/ml protein standard 500 ug/ml protein standard 5 uL 995 uL 0.082 500 ug/ml protein standard 10 uL 990 uL 0. 166 500 ug/ml protein standard 500 ug/ml protein standard 500 ug/ml protein standard 20 uL 980 uL 0.314 500 ug/ml protein standard 500 ug/ml protein standard 500 ug/ml protein standard 50 uL 950 uL 0.814 500 ug/mL 500 ug/mL protein standard 100 uL 900 uL 1.622 Unknown protein sam le Unknown protein sam le Unknown protein sam le 10 uL 990 uL 0. 132 Unknown protein sam le Unknown protein sam le Unknown protein sam le 100 uL 900 uL 1.362 Use this data to estimate the concentration of protein in the unknown sample as follows: Calculate the amount of protein added in each case for the 500 ug/mL standard samples. Calculate the concentration in mg/mL for each of these standard samples (hint: use CIVI = C2V2 to calculate the concentration of each standard after the water is added) Construct a graph of Absorbance vs. concentration for the standard samples (only use the standards here not your unknowns) Use the slope of this graph to back-calculate the concentration of the unknown protein sample in dilution Calculate the concentration of the original protein sample (undiluted) Example calculation for above: amount of protein added = volume protein added x concentration added = 5 NL x 500ug/mL = 5uL x 500ug/ 1000uL = 2.5ug concentration of standard sample = amount protein added / volume of sample = 2.5ug / (5uL + 995uL) = 2.5ug/ I000uL = 2.5ug/mL see excel section in back of lab manual equation of line: Abs = slope x [protein] so [protein] = Abs/slope = 0.082 / slope original concentration = [protein] from (d) x 1000/5 (I000uL in dultion / 5uL of stock) Show less
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