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How long to sonicate a total protain extraction? - (Aug/26/2008 )

Hi all,

Hope someone can help... I am trying to extract as much total protein from wheat seeds as possible. I am trying different buffers, with and without sonication. I have a probe sonicator to use, and my samples are in 300ul of buffer each time.

Has anyone any idea how long I should sonicate for, and at which intensity? I have no idea if it should be seconds or minutes.

Thanks for any help, I can't find any protocols anywhere!!

-Flour Power-

QUOTE (Flour Power @ Aug 27 2008, 01:17 AM)
Hi all,

Hope someone can help... I am trying to extract as much total protein from wheat seeds as possible. I am trying different buffers, with and without sonication. I have a probe sonicator to use, and my samples are in 300ul of buffer each time.

Has anyone any idea how long I should sonicate for, and at which intensity? I have no idea if it should be seconds or minutes.

Thanks for any help, I can't find any protocols anywhere!!

Are there any particular proteins you ar eafter? My old job was to prepare gliadins for a coelaic ELISA. We'd start from flour, extract the water-soluble fraction 2x (albumins etc) in PBS (1l per 100 g flour, mixed for 1 hr, then pelleted), then extract the gluten fraction (gliadin and glutenin) by resuspending the pellet in 40% EtOH for an hour. There ain't a whole lot left after that...

-swanny-

QUOTE (swanny @ Aug 27 2008, 07:11 AM)
QUOTE (Flour Power @ Aug 27 2008, 01:17 AM)
Hi all,

Hope someone can help... I am trying to extract as much total protein from wheat seeds as possible. I am trying different buffers, with and without sonication. I have a probe sonicator to use, and my samples are in 300ul of buffer each time.

Has anyone any idea how long I should sonicate for, and at which intensity? I have no idea if it should be seconds or minutes.

Thanks for any help, I can't find any protocols anywhere!!

Are there any particular proteins you ar eafter? My old job was to prepare gliadins for a coelaic ELISA. We'd start from flour, extract the water-soluble fraction 2x (albumins etc) in PBS (1l per 100 g flour, mixed for 1 hr, then pelleted), then extract the gluten fraction (gliadin and glutenin) by resuspending the pellet in 40% EtOH for an hour. There ain't a whole lot left after that...


So in your opinion would that be the best way to extract as much of the total protein as possible? I have at the moment been using SDS gel extraction buffers, and buffers containing TX-114. I assume these would extract water soluble proteins as good as pbs?

-Flour Power-

Because we were preparing antigen for a clinical assay, we had to show there were no significant amounts of other proteins, so I guess the two-stage process would remove as much protein as might be reasonably expected.

-swanny-