my protein belong to glycosyltransferase family 2 the catalytic domain adopt GT_A type fold , i used to purify with 20mM tris 8.0 100mM Nacl without additive, though the protein pI is 4.8. the crystal have got before was complex with UDP and glucose, that diffract at 5A, but now couldn't repeat the previous condition due to protein aggregation during purification,and also separate at different elution time behave like (monomer peak, dimer peak, and oligomer) from superdex 75 . then tried to purify with buffer supplemented with some additive like.
because the protein activity shows it's metal dependent specially (Mg+2 and Mn+2) do i need to add one of them during bacteria culture or to purification buffer ?
Dear Wafa,
One method to avoid aggregation is to change the buffer content such as pH, ionic strength and etc. In case this does not inhibit aggregation, an addition of some agents to the buffer such as EDTA, glycerol, arginine, glutamine, urea, and others may be helpful.
The following link describes ways to prevent aggegation:
http://www.sciencedirect.com/science/article/pii/S0003269703000599
Furthermore, protein solubility is largely dependent on the pH and ionic strength of the buffer used. Changing the pH to a value different from the isoelectrical point of the protein might help preventing aggregation. You can choose a pH above or under the isoelectrical point. For checking protein parameters please use the following link: http://web.expasy.org/protparam/.
Please note that the ionic strength of the buffer should be low and avoid any excess of salt.
Hoping this will be helpful,
Rafik
Dear Wafa,
One method to avoid aggregation is to change the buffer content such as pH, ionic strength and etc. In case this does not inhibit aggregation, an addition of some agents to the buffer such as EDTA, glycerol, arginine, glutamine, urea, and others may be helpful.
The following link describes ways to prevent aggegation:
http://www.sciencedirect.com/science/article/pii/S0003269703000599
Furthermore, protein solubility is largely dependent on the pH and ionic strength of the buffer used. Changing the pH to a value different from the isoelectrical point of the protein might help preventing aggregation. You can choose a pH above or under the isoelectrical point. For checking protein parameters please use the following link: http://web.expasy.org/protparam/.
Please note that the ionic strength of the buffer should be low and avoid any excess of salt.
Hoping this will be helpful,
Rafik
Dear Wafa,
If your enzyme requires Mg or Mn for activity, adding it to your purification buffers sounds like a good idea.
Good luck,
Pierre
Dear Guozhang,
You should avoid using surfactants having high CMC values.
Rafik
Hi Wafa,
as mentioned before, you can also try to add arginine to stabilize your protein. In general you should test for stable buffer solutions for your protein. When you have stable standard of your protein, you could test different pH values and different ionic strength. As an easy and quick method to analyze this I would suggest aggregation index (AI) measurements.
Kind regards
thanks Albert, could u plz suggest the concentration of arginine ? one more things my protein contain 6 cysteine residues do i need to add reducing agent ?
We have an unstable IgG molecule in house, which is stored in 50 mM phosphate and 350 mM arginine at appropiate pH. As mentioned before you should check which pH and ionic strength is suitable to stabilize your protein.
Regarding the cysteine residues: You could try adding a reducing agent like DTT to avoid aggregates formed by intermolecular disulfid bonds. Be aware that adding such a reducing agent can also unfold your protein, when the structure of your protein is stabilized by disulfide bonds.
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