A microbiologist performed a protein extraction from E.coli cells. After the ext

Question

A microbiologist performed a protein extraction from E.coli cells. After the extraction she did her best to separate the various impurities from the protein of interest in the sample. Her UV absorbance measurement gave her an absorbance of 1.493 at 280 nm and 0.143 at 260 nm. What is the approximate concentration in mg/mL) of her protein sample? Question 7 1.5 pts If you were asked to quantify vitamin B12 using UV-VIS, what would be an appropriate wavelength to use for your assay? Note: Report your answer in nm. This data e (also linked on the main page of Canvas) may be useful.

Explanation / Answer

1st Question

Approximate concentration can be obtained from the following corrected formula

Protein concentration (mg/ml) = 1.55A280 – 0.75A260

Where A280 is the absorbance at 280 nm and A260 is the absorbance at 260 nm

Therefore

concentration (mg/ml) = 1.55A280 – 0.75A260 = (1.55 x 1.493) - (0.75 x 0.143) = 2.314 - 0.107 = 2.207 mg/ml.

2nd question

Vitamin B12 has a maxima at 361 nm and also a band at near 550 nm. But the wave length at 361 nm is suitable for quantitative meassurements since molar extinction coefficient at 361 nm has a high value of 27,500cm-1/M.

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