Question

You are given a sample that has 1.3 mg/ml of purified protein. The molecular weight is 37,000 Daltons.

a) the absorbance in a 1cm cuvette is 0.93. calculate the molar extinction coefficient. Be sure to express your answer with correct units.

b)also calculate the extinction coefficient for concentration units of mg/ml, i.e. with units of cm^-1 (mg/ml)^-1

c) The sample is actually contaminated with 0.01 mg/ml DNA, but the protein concentration, 1.3 mg/ml is correct. explain how the contaminating DNA will affect your estimate of the extinction coefficient?

d) suggest a way in chich you could salvage this situation without doing any more experiments. that is, explain how you could estimate the extinction coefficient in spite of teh DNA contamination. what key inormation would you need to look up on the internet or in the library? carefully explain how you would calculate the extinction coefficient.

this is what I have done so far:

a) A=CLε

ε=0.93/(1.3mg/ml)*1cm

=.715(mg/ml)^-1Cm^-1

=.715(cm^-1) (ml/mg)(1L/1000ml)(1000mg/g)(37000g/mol)=26469(L/mol)=26469M^-1cm^-1

b) 26469M^-1cm^-1=26469cm^-1(L/mol)(1000ml/L)(1mol/37000g)(1g/1000mg)=.715(ml/mg)cm^-1=.715(mg/ml)^-1cm^-1

c) this would increase the absorbance, so it would make the extinction coefficient too high

d) ?

Answer 1

D) we can perform perform A260/280 absorbance using nano drop

for a pure DNA sample it is around 1.8 to 1.9

if the there is any protein in it the absorbance will be less than 1.8

280-nm Absorbance Method : Lab-Link

Most proteins contain one or more aromatic amino acids (tyrosine, tryptophan, and phenylalanine) that absorb light in the region 250 to 300 nm. The absorption spectrum of most proteins features a broad absorption usually centered around 275 to 285 nm, and spectrophotometric measurement at 280 nin is a frequently used method of estimating protein concentration. As will be discussed in more detail, a pure protein has a characteristic and defined extinction coefficient at 280 nrn that can, in pure solutions, be used to quantitate concentration very accurately based on spectrophotometric measurements. However, the extinction coefficient at 280 nin varies from approximately 0.5 to 1.5 for a 1mg/ml solution, making it difficult to obtain accurate estimates of total protein concentration for a, mixture of proteins (which, of course, is the normal situation encountered