which interferes with UV protein detection (at 280 nm/
260 nm) and may compromise separation by the generation
of complex detergent artifacts resulting in shifted retention
time for complexes. After each addition of HNN buffer and
the 5-min centrifugation, the residual volume should be
approximately 500 μL. Largely deviating volume indicates an
error. For example, if the volume is too low, the concentration
was not estimated correctly (BCA is not correct), or detergent
was added in a lower concentration than stated in the protocol.
If lysis conditions other than those outlined in this protocol are
applied, concentration time and exchange buffer volumes need
to be tested and optimized.
5. A higher level of precaution is necessary in the last step of
concentration when the sample reaches 8–9 mg/mL and protein precipitation is likely to occur. We recommend to stop the
concentration process at approximately 130/150 μL (approximately 7–8 mg/mL). Is it possible also to use a more conservative strategy which involves 1-min step centrifugation and
evaluation of the volume at every centrifugation step. We used
either strategy, and both have a high degree of reproducibility
across different samples. This is especially advisable if a sample
with an unknown behavior during the concentration step is
processed.
6. Refer to product manufacturing information for standard storage condition and usage. As an alternative, it is possible to
customize molecular curve calibration kit, using purified standards or polymers to cover the interested linear molecular
weight separation range.
7. The system setup should be configured to keep the peak broadening low. It is highly recommended to use PEEK capillaries
with small diameter (i.e., 0.005” ID), to minimize the length
of the flow path from column to fraction collector and to use a
sample loop with a volume smaller than 2% of total column
volume. The optimal flow rate should be optimized to avoid
sample diffusion but not increase system pressure and damage
the column resin.
8. A new column should be conditioned multiple times before
usage. We recommend to separate at least four times 700 μg of
whole cell lysate or the sample of interest if it is tissue. This step
saturates all surfaces, which tend to bind protein complexes,
and therefore highly increases reproducibility of separation
across replicates.
9. Particles, precipitates, and aggregates can damage the column;
always filter the standards (e.g., Spin-X, 0.45 μm cellulose
acetate filer, 4
C, 4000 Â g) and the samples (buffer exchange
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
289
260 nm) and may compromise separation by the generation
of complex detergent artifacts resulting in shifted retention
time for complexes. After each addition of HNN buffer and
the 5-min centrifugation, the residual volume should be
approximately 500 μL. Largely deviating volume indicates an
error. For example, if the volume is too low, the concentration
was not estimated correctly (BCA is not correct), or detergent
was added in a lower concentration than stated in the protocol.
If lysis conditions other than those outlined in this protocol are
applied, concentration time and exchange buffer volumes need
to be tested and optimized.
5. A higher level of precaution is necessary in the last step of
concentration when the sample reaches 8–9 mg/mL and protein precipitation is likely to occur. We recommend to stop the
concentration process at approximately 130/150 μL (approximately 7–8 mg/mL). Is it possible also to use a more conservative strategy which involves 1-min step centrifugation and
evaluation of the volume at every centrifugation step. We used
either strategy, and both have a high degree of reproducibility
across different samples. This is especially advisable if a sample
with an unknown behavior during the concentration step is
processed.
6. Refer to product manufacturing information for standard storage condition and usage. As an alternative, it is possible to
customize molecular curve calibration kit, using purified standards or polymers to cover the interested linear molecular
weight separation range.
7. The system setup should be configured to keep the peak broadening low. It is highly recommended to use PEEK capillaries
with small diameter (i.e., 0.005” ID), to minimize the length
of the flow path from column to fraction collector and to use a
sample loop with a volume smaller than 2% of total column
volume. The optimal flow rate should be optimized to avoid
sample diffusion but not increase system pressure and damage
the column resin.
8. A new column should be conditioned multiple times before
usage. We recommend to separate at least four times 700 μg of
whole cell lysate or the sample of interest if it is tissue. This step
saturates all surfaces, which tend to bind protein complexes,
and therefore highly increases reproducibility of separation
across replicates.
9. Particles, precipitates, and aggregates can damage the column;
always filter the standards (e.g., Spin-X, 0.45 μm cellulose
acetate filer, 4
C, 4000 Â g) and the samples (buffer exchange
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
289