niddk.nih.gov/clore/). For measurements at 214 nm, standard
amino acid extinction coefficients and a formula for computing ε
can be found in Ref. 43.
A simple protocol for a UV spectrum measurement is given
below:
1. Switch on the instrument and allow it to warm up for 20 min.
2. Record a spectrum with an empty sample compartment from
340 nm down to at least 240 nm and preferably 190 nm.
3. Insert a 1-cm quartz cuvette (see Note 5) and record a spectrum of the empty cuvette (in order to identify any possible
absorption of the cuvette itself or from contaminants at the
required wavelengths).
4. Add buffer and measure the absorbance of cuvette plus buffer.
This should be exactly the same buffer that used to prepare the
protein solution (see Note 6). The absorbance will go up at low
wavelengths as carbonyl and amide bonds found in many buffers as well as salts absorb here; DTT and β-mercaptoethanol
absorb at higher wavelengths (see Note 7). It is best that the
buffer is transparent at the required wavelengths or, if there is
some absorbance, make sure that the absorbance is much smaller than the limit of the instrument. If not, use a lower concentration or less absorbent buffer.
5. Re-measure the buffer to make sure that it gives consistent
results. This test checks whether the lamp is warm and excludes
lamp intensity fluctuations.
6. Run a baseline/blank with buffer (this will be subtracted from
future measurements to produce a spectrum for the sample
alone).
7. Run a spectrum with the protein solution and inspect the
spectrum shape: check that the maximum absorption is below
the high absorption limit of the instrument. If this is not the
case, dilute the protein solution with buffer and repeat the
measurement.
8. Calculate AI and the A 260 /A 280 ratio (Subheading 1.1). The
AI should be below 2 and the A 260 /A 280 ratio below 0.6 to be
sure that the sample is sufficiently free of aggregation and
nucleic acid contamination. If these conditions are met, use
the A 280 , A 214 , or A 205 value and the corresponding extinction
coefficient to calculate the concentration of the protein solution in the cuvette (see Note 6).
Bear in mind that both the measurement and the extinction
coefficient calculation will always have some error and the true
concentration is frequently 5% different from the value estimated
by UV measurement. However, no other method is more accurate
22
Bertrand Raynal et al.
amino acid extinction coefficients and a formula for computing ε
can be found in Ref. 43.
A simple protocol for a UV spectrum measurement is given
below:
1. Switch on the instrument and allow it to warm up for 20 min.
2. Record a spectrum with an empty sample compartment from
340 nm down to at least 240 nm and preferably 190 nm.
3. Insert a 1-cm quartz cuvette (see Note 5) and record a spectrum of the empty cuvette (in order to identify any possible
absorption of the cuvette itself or from contaminants at the
required wavelengths).
4. Add buffer and measure the absorbance of cuvette plus buffer.
This should be exactly the same buffer that used to prepare the
protein solution (see Note 6). The absorbance will go up at low
wavelengths as carbonyl and amide bonds found in many buffers as well as salts absorb here; DTT and β-mercaptoethanol
absorb at higher wavelengths (see Note 7). It is best that the
buffer is transparent at the required wavelengths or, if there is
some absorbance, make sure that the absorbance is much smaller than the limit of the instrument. If not, use a lower concentration or less absorbent buffer.
5. Re-measure the buffer to make sure that it gives consistent
results. This test checks whether the lamp is warm and excludes
lamp intensity fluctuations.
6. Run a baseline/blank with buffer (this will be subtracted from
future measurements to produce a spectrum for the sample
alone).
7. Run a spectrum with the protein solution and inspect the
spectrum shape: check that the maximum absorption is below
the high absorption limit of the instrument. If this is not the
case, dilute the protein solution with buffer and repeat the
measurement.
8. Calculate AI and the A 260 /A 280 ratio (Subheading 1.1). The
AI should be below 2 and the A 260 /A 280 ratio below 0.6 to be
sure that the sample is sufficiently free of aggregation and
nucleic acid contamination. If these conditions are met, use
the A 280 , A 214 , or A 205 value and the corresponding extinction
coefficient to calculate the concentration of the protein solution in the cuvette (see Note 6).
Bear in mind that both the measurement and the extinction
coefficient calculation will always have some error and the true
concentration is frequently 5% different from the value estimated
by UV measurement. However, no other method is more accurate
22
Bertrand Raynal et al.