Exactly. Without a picture of SEC profile it's even difficult to decipher the
meaning of "extremely asymmetric peak shape".
Beside that, one domain or multi-domain protein, extended N-term or C-term
loops containing constructs, column, load (weight & vol) and flow rate - any
one of these could
Probably a good idea to share an image :) worth many words...
Artem
On Wed, Dec 9, 2020, 9:17 AM wrote:
> Dear All
> There is a 43kd protein purified via Ni-chelating affinity
> chromatography, anion exchange chromatography and gel filtration
> chromatography in sequence. However the
It would be sensible to analyse the state of your purified protein further.
* (SEC in reasonable salt conc containing buffer – this is standard
practice)
* Analytical ion exchange of purified protein - are there different states?
* SEC (in reasonable buffer) in line with MALS – is
Hello, I agree with Roger, you should definitely try to increase the salt
concentration to get rid of non specific binding impurities. And if that
doesn't work, you can try purifying your protein under denaturing
conditions by adding one refolding step in the column.
Good luck,
Javier
On Wed, Dec
Salt concentrations less than 100 mM can lead to nonspecific adsorption to
the gel exclusion media, potentially leading to band broadening, and
delayed elution. Overloading gel exclusion columns (more than 2-4% Vt) can
also lead to elution band artifacts. Check these issues first.
Roger Rowlett
