account their expected oligomeric state. A similar approach is
used to determine the value of (dn/dc) nucleic acid ; values
between 0.16 and 0.19 ml/g have been used, with smaller
values associated with longer nucleic acids [1, 2, 14, 20,
23]. Since the value of (dn/dc) nucleic acid varies depending on
the length and secondary structure of the nucleic acid and
buffer conditions, it is imperative to perform SEC/MALS
analysis of the exact DNA or RNA, of known sequence and
M w , that is used for complex formation and validate the refractive index increment for this particular piece of nucleic acid in
the buffer conditions used for SEC/MALS analysis.
12. It is critically important to analyze the samples at various concentrations to determine whether the observed M w is affected
by changes in concentration. If sample’s amount is limited, the
eluting material can be collected as eluting fractions and
re-analyzed at lower concentrations. Both M w and UV/RI
ratio should be monitored as a function of concentration to
establish whether the complex is stable like observed for E.r.
RT domain (Fig. 1b, c and Table 1) or experiencing dynamic
dissociation process like observed for FIR protein (Fig. 2b, c
and Table 2) before reporting stoichiometry; M w should be
within 10% and UV/RI ratio within 20% of predicted values.
References
1. Zhao C, Pyle AM (2016) Crystal structures of a
group II intron maturase reveal a missing link
in spliceosome evolution. Nat Struct Mol Biol
23(6):558–565. https://doi.org/10.1038/
nsmb.3224
2. Crichlow GV, Zhou H, Hsiao HH, Frederick
KB, Debrosse M, Yang Y, Folta-Stogniew EJ,
Chung HJ, Fan C, De la Cruz EM, Levens D,
Lolis E, Braddock D (2008) Dimerization of
FIR upon FUSE DNA binding suggests a
mechanism of c-myc inhibition. EMBO J 27
(1):277–289.
https://doi.org/10.1038/sj.
emboj.7601936
3. Wyatt PJ (1993) Light scattering and the absolute characterization of macromolecules. Anal
Chim Acta 272(1):1–40. https://doi.org/10.
1016/0003-2670(93)80373-S
4. Takagi T (1990) Application of low-angle laser
light scattering detection in the field of biochemistry: review of recent progress. J Chromatogr A 506:409–416. https://doi.org/10.
1016/S0021-9673(01)91596-1
5. Wen J, Arakawa T, Philo JS (1996) Sizeexclusion chromatography with on-line lightscattering, absorbance, and refractive index
detectors for studying proteins and their
interactions. Anal Biochem 240(2):155–166.
https://doi.org/10.1006/abio.1996.0345
6. Folta-Stogniew E, Williams KR (1999) Determination of molecular masses of proteins in
solution: implementation of an HPLC size
exclusion chromatography and laser light scattering service in a core laboratory. J Biomol
Tech 10(2):51–63
7. Minton AP (2016) Recent applications of light
scattering measurement in the biological and
biopharmaceutical sciences. Anal Biochem
501:4–22.
https://doi.org/10.1016/j.ab.
2016.02.007
8. Mogridge J (2015) Using light scattering to
determine the stoichiometry of protein complexes. Methods Mol Biol 1278:233–238.
https://doi.org/10.1007/978-1-4939-24257_14
9. Gupta K, Curtis Joseph E, Krueger S,
Hwang Y, Cherepanov P, Bushman
Frederic D, Van Duyne Gregory D (2012)
Solution conformations of prototype foamy
virus integrase and its stable synaptic complex
with U5 viral DNA. Structure 20
(11):1918–1928. https://doi.org/10.1016/j.
str.2012.08.023
394
Ewa Folta-Stogniew
used to determine the value of (dn/dc) nucleic acid ; values
between 0.16 and 0.19 ml/g have been used, with smaller
values associated with longer nucleic acids [1, 2, 14, 20,
23]. Since the value of (dn/dc) nucleic acid varies depending on
the length and secondary structure of the nucleic acid and
buffer conditions, it is imperative to perform SEC/MALS
analysis of the exact DNA or RNA, of known sequence and
M w , that is used for complex formation and validate the refractive index increment for this particular piece of nucleic acid in
the buffer conditions used for SEC/MALS analysis.
12. It is critically important to analyze the samples at various concentrations to determine whether the observed M w is affected
by changes in concentration. If sample’s amount is limited, the
eluting material can be collected as eluting fractions and
re-analyzed at lower concentrations. Both M w and UV/RI
ratio should be monitored as a function of concentration to
establish whether the complex is stable like observed for E.r.
RT domain (Fig. 1b, c and Table 1) or experiencing dynamic
dissociation process like observed for FIR protein (Fig. 2b, c
and Table 2) before reporting stoichiometry; M w should be
within 10% and UV/RI ratio within 20% of predicted values.
References
1. Zhao C, Pyle AM (2016) Crystal structures of a
group II intron maturase reveal a missing link
in spliceosome evolution. Nat Struct Mol Biol
23(6):558–565. https://doi.org/10.1038/
nsmb.3224
2. Crichlow GV, Zhou H, Hsiao HH, Frederick
KB, Debrosse M, Yang Y, Folta-Stogniew EJ,
Chung HJ, Fan C, De la Cruz EM, Levens D,
Lolis E, Braddock D (2008) Dimerization of
FIR upon FUSE DNA binding suggests a
mechanism of c-myc inhibition. EMBO J 27
(1):277–289.
https://doi.org/10.1038/sj.
emboj.7601936
3. Wyatt PJ (1993) Light scattering and the absolute characterization of macromolecules. Anal
Chim Acta 272(1):1–40. https://doi.org/10.
1016/0003-2670(93)80373-S
4. Takagi T (1990) Application of low-angle laser
light scattering detection in the field of biochemistry: review of recent progress. J Chromatogr A 506:409–416. https://doi.org/10.
1016/S0021-9673(01)91596-1
5. Wen J, Arakawa T, Philo JS (1996) Sizeexclusion chromatography with on-line lightscattering, absorbance, and refractive index
detectors for studying proteins and their
interactions. Anal Biochem 240(2):155–166.
https://doi.org/10.1006/abio.1996.0345
6. Folta-Stogniew E, Williams KR (1999) Determination of molecular masses of proteins in
solution: implementation of an HPLC size
exclusion chromatography and laser light scattering service in a core laboratory. J Biomol
Tech 10(2):51–63
7. Minton AP (2016) Recent applications of light
scattering measurement in the biological and
biopharmaceutical sciences. Anal Biochem
501:4–22.
https://doi.org/10.1016/j.ab.
2016.02.007
8. Mogridge J (2015) Using light scattering to
determine the stoichiometry of protein complexes. Methods Mol Biol 1278:233–238.
https://doi.org/10.1007/978-1-4939-24257_14
9. Gupta K, Curtis Joseph E, Krueger S,
Hwang Y, Cherepanov P, Bushman
Frederic D, Van Duyne Gregory D (2012)
Solution conformations of prototype foamy
virus integrase and its stable synaptic complex
with U5 viral DNA. Structure 20
(11):1918–1928. https://doi.org/10.1016/j.
str.2012.08.023
394
Ewa Folta-Stogniew