based on weight and the volume of buffer used to dissolve
(Mr 628 Da).
4. Measuring protein concentration using absorbance requires a
careful and methodical approach. Instruments must be blanked
with appropriate solvent, and the measured absorbance should
be within a reliable absorbance range and be corrected for any
scattering contributions determined by measuring the full
spectrum including longer wavelength data where the protein
should not absorb (see Note 10). A suitable lysozyme solution
for this test reaction is ~40 μM (having absorbance of
~1.5 cm
À1 that can be measured in a 1 cm cuvette or at
1 mm which is common for micro volume measurement such
as on a Nanodrop). For the iTC200 instrument and using the
supplied loading syringe, a volume of ~350 μL is required.
5. The lysozyme should be loaded into the ITC cell using the
syringe supplied with the instrument and following protocols
recommended by the manufacturer to produce a total fill without any trapped air. It is also common that users develop their
own variants of standard loading protocols and these “local
practices” are also useful if they have a proven track record.
The loading process is the tricky part of performing an ITC
experiment and so it is worth practicing this with test reactions
(see Subheading 3.1, step 3 to evaluate success). There is an
established history that degassing the solutions should help
with loading but there is the risk that using low pressure can
lead to evaporation and changes in concentration. Worse still is
the possibility of denaturation of protein samples at the solution surface if stirring is used. So degassing should be done
with care and the concentrations and integrity of material
checked afterwards. What seems more useful in loading is to
ensure that the ITC cell is spotlessly clean which is best
achieved by storing the instrument with dilute laboratory
detergent in the cells when not in use. This approach can be
combined with more vigorous treatments such as incubating
the cells at 50
C overnight in detergent or the use of strong
acid or alkali solutions (taking great care to comply with the
detailed chemical compatibility of the cell construction that will
be supplied by the manufacturer).
6. The tri-acetyl glucosamine should be prepared and loaded into
the syringe as instructed in the instrument manual at ~20-fold
higher concentration than the lysozyme (~800 μM). For the
iTC200, this requires ~70 μL.
7. Both of the required volumes are larger than the active cell
volume and syringe capacity (~200 μL and 40 μL, respectively
for the iTC200) and some solution is left after loading. These
small aliquots can be used to check concentration and sample
integrity (at least for the lysozyme) since they most closely
resemble the true experimental solutions being used.
140
Christopher M. Johnson
(Mr 628 Da).
4. Measuring protein concentration using absorbance requires a
careful and methodical approach. Instruments must be blanked
with appropriate solvent, and the measured absorbance should
be within a reliable absorbance range and be corrected for any
scattering contributions determined by measuring the full
spectrum including longer wavelength data where the protein
should not absorb (see Note 10). A suitable lysozyme solution
for this test reaction is ~40 μM (having absorbance of
~1.5 cm
À1 that can be measured in a 1 cm cuvette or at
1 mm which is common for micro volume measurement such
as on a Nanodrop). For the iTC200 instrument and using the
supplied loading syringe, a volume of ~350 μL is required.
5. The lysozyme should be loaded into the ITC cell using the
syringe supplied with the instrument and following protocols
recommended by the manufacturer to produce a total fill without any trapped air. It is also common that users develop their
own variants of standard loading protocols and these “local
practices” are also useful if they have a proven track record.
The loading process is the tricky part of performing an ITC
experiment and so it is worth practicing this with test reactions
(see Subheading 3.1, step 3 to evaluate success). There is an
established history that degassing the solutions should help
with loading but there is the risk that using low pressure can
lead to evaporation and changes in concentration. Worse still is
the possibility of denaturation of protein samples at the solution surface if stirring is used. So degassing should be done
with care and the concentrations and integrity of material
checked afterwards. What seems more useful in loading is to
ensure that the ITC cell is spotlessly clean which is best
achieved by storing the instrument with dilute laboratory
detergent in the cells when not in use. This approach can be
combined with more vigorous treatments such as incubating
the cells at 50
C overnight in detergent or the use of strong
acid or alkali solutions (taking great care to comply with the
detailed chemical compatibility of the cell construction that will
be supplied by the manufacturer).
6. The tri-acetyl glucosamine should be prepared and loaded into
the syringe as instructed in the instrument manual at ~20-fold
higher concentration than the lysozyme (~800 μM). For the
iTC200, this requires ~70 μL.
7. Both of the required volumes are larger than the active cell
volume and syringe capacity (~200 μL and 40 μL, respectively
for the iTC200) and some solution is left after loading. These
small aliquots can be used to check concentration and sample
integrity (at least for the lysozyme) since they most closely
resemble the true experimental solutions being used.
140
Christopher M. Johnson