protein degardation during gel filteration
Posted 24 June 2009 - 10:33 PM
Posted 25 June 2009 - 07:54 AM
is the g-100 column fresh packed? with fresh g-100?
we had a situation where we reused a gel filtration column and our protein was degraded. the column had become contaminated with a protease from a previous sample.
as a side note, you may need to increase the ionic strength of your buffer to prevent nonspecific binding of your protein to the sepharose.
genius does what it must
i do what i get paid to do
Posted 27 June 2009 - 09:28 PM
1. Use 500 mM NaCl and 5-10 mM imidazole in the wash buffer along with tris/phosphate and glycerol and wash the protein bound to Ni-NTA beads with this wash buffer. THis will take care of non specific proteins.
2. Use ice cold buffers. This will take care of degradation. Alternatively, use protease inhibitors.
Hope this helps.