I need a sample that contains aggregates for testing my chromatography process.
It will depend on the actual protein of course but a good way to start is to change the pH to pI of the protein. If that does not work, conditions that favor the molten globule state (low concentrations of denaturant (1-2M GuHCl or urea) may help. Adjusting the ionic strength may also favor aggregation. Which way to adjust depends on the protein. Generally, from a consideration of surface tension effects only, high ionic strength favors aggregation if a hydophobic patch is exposed in the native state, low ionic strength favors aggregation if the hydrophobic patch is exposed only when the protein is partially unfolded.
It will depend on the actual protein of course but a good way to start is to change the pH to pI of the protein. If that does not work, conditions that favor the molten globule state (low concentrations of denaturant (1-2M GuHCl or urea) may help. Adjusting the ionic strength may also favor aggregation. Which way to adjust depends on the protein. Generally, from a consideration of surface tension effects only, high ionic strength favors aggregation if a hydophobic patch is exposed in the native state, low ionic strength favors aggregation if the hydrophobic patch is exposed only when the protein is partially unfolded.
Rightly said by Jeffrey. Since you have solution of unfolded proteins, you can either neutralize surface charge, so that monomers interact to form aggregates or you can also try heating at >65 degree Celsius as in some cases that will lead to amyloid fiber formation by peptides.
You already have a solution of unfolded proteins and they do not aggregate? That's unusual. In addition to seconding Jeffrey's sound advice, I would try concentrating the protein, or switching to a different model protein altogether. And if there is a chaotrope present, remove it.
Hello,
The easiest way to aggregate is to heat protein. 40C for 2-3 hours should work.
Chemical way is to deamidate in basic (borate or carbonate) buffer at higher temp such 37C for 24 hrs. This will add some charges and at low salt proteins will aggregate.
You can also do molecular crowding with PEG solution (titrate %) or add isopropanol to up to 30%.
Regards,
Galina
Sorry, I made a typo, the heat inactivation starts from 50C and up, the 2-3 hrs incubation is for 70C, not 40C. Or you can titrate time at different temperatures - 50, 60, 70.
Regards,
Galina
Heat protein samples at pH range nearing to its PI at ~65C for few hours.
or else heating at very low pH
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