and finer increments of pH (and alternative buffers for the beneficial pH range) to enable optimization of solubility and stability.
A typical strategy would be to combine screening by DLS for
homogeneity and solubility and a DSF method for thermostability.
Having identified solution conditions giving good results using
these methods, further investigation by CD (and DCS) should be
used to confirm structural integrity in the conditions.
3 Notes
1. Whereas the presence of cofactors can confound concentration
determination, a UV/Vis spectrum can also enable a fast and
efficient batch-to-batch comparison relating to cofactor content and thus evaluate functional equivalence of different samples. Flavoproteins, for example, contain a flavin as a cofactor,
and a UV/Vis spectrum of such a sample provides information
on which flavin is bound (flavin mononucleotide (FMD) or
flavin adenine dinucleotide (FAD)), how much flavin is present, and if the flavin is in the reduced or oxidized state [46].
2. In a PA concentration range of 10–19% containing 0.1% (w/v)
SDS, there is a logarithmic relationship between MW and the
migration distance of the SDS-polypeptide micelles, which may
be exploited to determine more accurate estimates of the
masses of proteins. The relative migration distance is determined as the ratio of the distance of the band of the protein
to the distance of the buffer (which corresponds approximately
to the distance covered by bromophenol blue in the sample
buffer) from the beginning of the resolving gel. Then, a standard curve is generated by plotting the relative migration distances of the proteins of the size marker semi-logarithmically
against their known molecular weights, followed by a leastsquares fit of these data points. This standard curve can be
used to estimate the molecular weights of the unknown
proteins.
3. The European consensus on protein QC can be found on the
websites of the European networks P4EU (https://p4eu.org/
protein-quality-standard-pqs)
and
ARBRE-MOBIEU
(https://arbre-mobieu.eu/guidelines-on-protein-qualitycontrol).
4. In order to “polish” the purified protein and, in particular, to
remove protein aggregates, the last step of the purification
process should always be a SEC run. It is often necessary to
further concentrate protein samples for their downstream
applications. Unfortunately, this process, which is often done
by ultrafiltration using spin concentrators or precipitation/
resolublization protocols, very frequently induces aggregation.
40
Bertrand Raynal et al.
A typical strategy would be to combine screening by DLS for
homogeneity and solubility and a DSF method for thermostability.
Having identified solution conditions giving good results using
these methods, further investigation by CD (and DCS) should be
used to confirm structural integrity in the conditions.
3 Notes
1. Whereas the presence of cofactors can confound concentration
determination, a UV/Vis spectrum can also enable a fast and
efficient batch-to-batch comparison relating to cofactor content and thus evaluate functional equivalence of different samples. Flavoproteins, for example, contain a flavin as a cofactor,
and a UV/Vis spectrum of such a sample provides information
on which flavin is bound (flavin mononucleotide (FMD) or
flavin adenine dinucleotide (FAD)), how much flavin is present, and if the flavin is in the reduced or oxidized state [46].
2. In a PA concentration range of 10–19% containing 0.1% (w/v)
SDS, there is a logarithmic relationship between MW and the
migration distance of the SDS-polypeptide micelles, which may
be exploited to determine more accurate estimates of the
masses of proteins. The relative migration distance is determined as the ratio of the distance of the band of the protein
to the distance of the buffer (which corresponds approximately
to the distance covered by bromophenol blue in the sample
buffer) from the beginning of the resolving gel. Then, a standard curve is generated by plotting the relative migration distances of the proteins of the size marker semi-logarithmically
against their known molecular weights, followed by a leastsquares fit of these data points. This standard curve can be
used to estimate the molecular weights of the unknown
proteins.
3. The European consensus on protein QC can be found on the
websites of the European networks P4EU (https://p4eu.org/
protein-quality-standard-pqs)
and
ARBRE-MOBIEU
(https://arbre-mobieu.eu/guidelines-on-protein-qualitycontrol).
4. In order to “polish” the purified protein and, in particular, to
remove protein aggregates, the last step of the purification
process should always be a SEC run. It is often necessary to
further concentrate protein samples for their downstream
applications. Unfortunately, this process, which is often done
by ultrafiltration using spin concentrators or precipitation/
resolublization protocols, very frequently induces aggregation.
40
Bertrand Raynal et al.