7. Set up a program containing 10–30 acquisitions with an acquisition time of 2–10 s per sample. The appropriate combination
of number and time depends on whether or not dust or other
larger particles disturb the measurement. Specifically, use a
large number of short acquisitions if individual acquisitions
give erratic results. Measurements should be done in technical
triplicates using automatic laser power and detector
attenuation.
8. If using a multiwell plate DLS instrument with a camera, an
image of each well should be captured, as this may help in
interpretation of data analysis by visualizing artifacts (e.g.,
phase separation, bubbles).
9. During data analysis, first the data from buffer alone should be
checked to confirm that the buffer itself does not contain large
particles. Figure 3a shows the typical autocorrelation curve for
a clean buffer with a weak intensity of the autocorrelation
curve.
10. Check the shape of the autocorrelation curve for the protein
(Fig. 3b, inset). Check the fit and the error of the measurement. The presence of aggregates is indicated if particles with
R h of more than 10 times that of the expected size of the
monomer can be observed (Fig. 3b).
11. If the protein is not detectable due to the presence of too much
aggregate or other artifacts, the sample should be centrifuged
using a benchtop centrifuge (15 min at full speed). Remeasure
the concentration after centrifugation as protein content will
be reduced.
2.2.4 Protein Intact Mass
by MALDI-TOF Mass
Spectrometry
Protein intact mass can be assessed by either ESI or MALDI MS
approaches. Here, we will focus on MALDI-TOF MS. During
sample preparation for a MALDI experiment, a droplet of the
target protein:matrix solution is deposited onto a MALDI plate
and the solvent is evaporated to allow co-crystallization. There are
several ways to prepare deposits, varying in matrix concentration,
solvent, method of crystallization, etc. The “dried droplet” method
is the most commonly used technique because it is extremely fast
and simple to implement.
A simple protocol for intact mass by MALDI is given below:
1. Prepare the protein sample. For a typical 15-kDa protein, 1μL
at 0.1 mg/mL is sufficient, and for a 150 kDa protein, around
1 mg/mL is recommended. These concentrations are acceptable for protein samples in phosphate-buffered saline (PBS)type buffers. If your protein sample buffer is supplemented
with more than 1% (v/v) glycerol or detergent or in high salt,
the protein should be prepared at a 10Â higher concentration
and then diluted with water to reach the required
Protein Quality Control
29
of number and time depends on whether or not dust or other
larger particles disturb the measurement. Specifically, use a
large number of short acquisitions if individual acquisitions
give erratic results. Measurements should be done in technical
triplicates using automatic laser power and detector
attenuation.
8. If using a multiwell plate DLS instrument with a camera, an
image of each well should be captured, as this may help in
interpretation of data analysis by visualizing artifacts (e.g.,
phase separation, bubbles).
9. During data analysis, first the data from buffer alone should be
checked to confirm that the buffer itself does not contain large
particles. Figure 3a shows the typical autocorrelation curve for
a clean buffer with a weak intensity of the autocorrelation
curve.
10. Check the shape of the autocorrelation curve for the protein
(Fig. 3b, inset). Check the fit and the error of the measurement. The presence of aggregates is indicated if particles with
R h of more than 10 times that of the expected size of the
monomer can be observed (Fig. 3b).
11. If the protein is not detectable due to the presence of too much
aggregate or other artifacts, the sample should be centrifuged
using a benchtop centrifuge (15 min at full speed). Remeasure
the concentration after centrifugation as protein content will
be reduced.
2.2.4 Protein Intact Mass
by MALDI-TOF Mass
Spectrometry
Protein intact mass can be assessed by either ESI or MALDI MS
approaches. Here, we will focus on MALDI-TOF MS. During
sample preparation for a MALDI experiment, a droplet of the
target protein:matrix solution is deposited onto a MALDI plate
and the solvent is evaporated to allow co-crystallization. There are
several ways to prepare deposits, varying in matrix concentration,
solvent, method of crystallization, etc. The “dried droplet” method
is the most commonly used technique because it is extremely fast
and simple to implement.
A simple protocol for intact mass by MALDI is given below:
1. Prepare the protein sample. For a typical 15-kDa protein, 1μL
at 0.1 mg/mL is sufficient, and for a 150 kDa protein, around
1 mg/mL is recommended. These concentrations are acceptable for protein samples in phosphate-buffered saline (PBS)type buffers. If your protein sample buffer is supplemented
with more than 1% (v/v) glycerol or detergent or in high salt,
the protein should be prepared at a 10Â higher concentration
and then diluted with water to reach the required
Protein Quality Control
29