3.4 Determination
of the Molecular
Weight of the Sample
Protein, Nucleic Acid,
and Protein–Nucleic
Acid Complex
1. Filter all samples through a 0.22 μm low-protein-binding filter
(see Note 6).
2. Inject an appropriate amount of sample (see Table 4 for optimal
amounts of sample) and record signals from all three detectors.
3. Inject all samples in a series of three or more concentrations
spanning at least one order of magnitude (see Note 12).
4. Baseline-correct the signal from all three detectors.
5. Calculate molecular weights for all samples analyzed.
3.5 Determination
of Monodispersity
1. Compute (dn/dc) complex as a weight average of (dn/dc) protein
and (dn/dc) nucleic acid based on proposed stoichiometry (see
Note 11).
2. Use the (dn/dc) complex during processing of the SEC/MALS
data using ASTRA software (see Note 11).
3. Generate a molar mass distribution plot for all samples
analyzed, i.e., protein alone, nucleic acid alone, and the complex (examples of such plots for E.r.RT domain are shown in
Fig. 1a, b and for FIR protein in Fig. 2a, b).
4. Check the distribution of M w across the eluting peak; for a
monodisperse sample, it should vary by no more than Æ5%
for the middle portion of the peak that is above half-height
(thus is not significantly affected by band-broadening effects).
5. Check whether the M w varies with changes in the concentration of the injected sample by injecting the same volume of a
sample that is tenfold diluted from the original sample; for
monodisperse samples, the M w measured for the apex portion
of the eluting peak should be concentration independent.
3.6 Determination
of Stoichiometry, i.e.,
Protein to Nucleic Acid
Ratio in the Complex
1. Compute the M w for the possible stoichiometries. If the
observed M w of the complex is concentration independent
and within 10% of the M w for only one of the stoichiometries
proposed for the complex, the stoichiometry is computed
based on M w alone (like for E.r. RT + D4A RNA complex;
Fig. 1b, c).
2. If the observed M w varies with concentration, plot the M w as a
function of concentration and validate that the observed
dependence is close to a plateau (Fig. 2c); if the observed M w
is within 10% of only one of the M w proposed for the possible
stoichiometries, the complex stoichiometry is computed based
on M w alone.
3. If the stoichiometry cannot be determined based on the M w
alone (like for FIR-FUSE DNA complex; Table 3), compute
the volume of UV and RI peaks generated during SEC/MALS
analyses of the individual components of the complex, i.e., the
samples: protein alone and nucleic acid alone. Compute the
390
Ewa Folta-Stogniew
of the Molecular
Weight of the Sample
Protein, Nucleic Acid,
and Protein–Nucleic
Acid Complex
1. Filter all samples through a 0.22 μm low-protein-binding filter
(see Note 6).
2. Inject an appropriate amount of sample (see Table 4 for optimal
amounts of sample) and record signals from all three detectors.
3. Inject all samples in a series of three or more concentrations
spanning at least one order of magnitude (see Note 12).
4. Baseline-correct the signal from all three detectors.
5. Calculate molecular weights for all samples analyzed.
3.5 Determination
of Monodispersity
1. Compute (dn/dc) complex as a weight average of (dn/dc) protein
and (dn/dc) nucleic acid based on proposed stoichiometry (see
Note 11).
2. Use the (dn/dc) complex during processing of the SEC/MALS
data using ASTRA software (see Note 11).
3. Generate a molar mass distribution plot for all samples
analyzed, i.e., protein alone, nucleic acid alone, and the complex (examples of such plots for E.r.RT domain are shown in
Fig. 1a, b and for FIR protein in Fig. 2a, b).
4. Check the distribution of M w across the eluting peak; for a
monodisperse sample, it should vary by no more than Æ5%
for the middle portion of the peak that is above half-height
(thus is not significantly affected by band-broadening effects).
5. Check whether the M w varies with changes in the concentration of the injected sample by injecting the same volume of a
sample that is tenfold diluted from the original sample; for
monodisperse samples, the M w measured for the apex portion
of the eluting peak should be concentration independent.
3.6 Determination
of Stoichiometry, i.e.,
Protein to Nucleic Acid
Ratio in the Complex
1. Compute the M w for the possible stoichiometries. If the
observed M w of the complex is concentration independent
and within 10% of the M w for only one of the stoichiometries
proposed for the complex, the stoichiometry is computed
based on M w alone (like for E.r. RT + D4A RNA complex;
Fig. 1b, c).
2. If the observed M w varies with concentration, plot the M w as a
function of concentration and validate that the observed
dependence is close to a plateau (Fig. 2c); if the observed M w
is within 10% of only one of the M w proposed for the possible
stoichiometries, the complex stoichiometry is computed based
on M w alone.
3. If the stoichiometry cannot be determined based on the M w
alone (like for FIR-FUSE DNA complex; Table 3), compute
the volume of UV and RI peaks generated during SEC/MALS
analyses of the individual components of the complex, i.e., the
samples: protein alone and nucleic acid alone. Compute the
390
Ewa Folta-Stogniew