Protein precipitation compatible with Sucrose gradients? - What precipitation protocol wil function with a high % of sucrose? (Nov/16/2007 )
Hello everyone, I've been running a lot of sucrose gradients lately for subsequent analysis on SDS-Page and MS-MS. Sadly it seems that my preferred acetone precipitation protocol also precipitates the sucrose due to the very low solubility in acetone and gives a viscous sludge . So I'm wondering about TCA or TCA/DOC. Has anyone used these protocols with a sucrose concentration of around 50%?
On the other side of things I am thinking of diluting my acetone down to 80% and seeing if that allows the Sucrose to stay soluble but I'm uncertain of how this would affect precipitation.
Any suggestions? I would prefer not to dilute my samples since I'm working with a lot of fractions so any hints are very welcome!
Best regards
Erik
Get some ultrafiltration fiter units for microcentrifuge with a MW cutoff 3000. I think these will work great for you.
or, you could try ammonium sulfate precipitation.
I'm a bit uncertain about the concentration of Ammonium Sulfate that will give a total precipitation of protein though.
It's no problem for me to achieve a concentration of around 90% ammonium sulfate and still have a simple workflow. Would this be enough to get a total protein pellet?
A good idea, I'll look into it.
I'm a bit uncertain about the concentration of Ammonium Sulfate that will give a total precipitation of protein though.
It's no problem for me to achieve a concentration of around 90% ammonium sulfate and still have a simple workflow. Would this be enough to get a total protein pellet?
Typically no. The efficiency is protein type and protein concentration dependent.