Protein pellet won't resuspend in buffer following acetone precipitation

Hey gang,

I just completed a nuclear protein extraction. I tried to resuspend my pellet after acetone precipitation in the buffer we use but the pellet won't redissolve. I tried vortexing and evening using a syringe but nothing's working, there's still particles that won't go into solution.

I need the samples for Western blotting and still need to quantify my samples as well.

I was thinking of leaving the samples at room temperature (they're on ice right now), maybe that would help?

Any suggestions would be appreciated.

Thanx,

RS

QUOTE (Rohita @ Feb 8 2007, 07:10 PM)

Hey gang,

I just completed a nuclear protein extraction. I tried to resuspend my pellet after acetone precipitation in the buffer we use but the pellet won't redissolve. I tried vortexing and evening using a syringe but nothing's working, there's still particles that won't go into solution.

I need the samples for Western blotting and still need to quantify my samples as well.

I was thinking of leaving the samples at room temperature (they're on ice right now), maybe that would help?

Any suggestions would be appreciated.

Thanx,

RS

often scaffolding proteins are difficult to resolve after precipitation; as suggested most are to dissolve in Laemmli sample buffer; in this case, quantification is possible after gel-run with f.i. CBB staining; the disadvantage is the second run for Wb;

another possibility is to trying to dissolve pellets in a very low amount of DMSO or DMF;

sometimes recommended sonfication protocools may be harmful to proteins

QUOTE (The Bearer @ Feb 9 2007, 10:25 AM)

QUOTE (Rohita @ Feb 8 2007, 07:10 PM)

Hey gang,

I just completed a nuclear protein extraction. I tried to resuspend my pellet after acetone precipitation in the buffer we use but the pellet won't redissolve. I tried vortexing and evening using a syringe but nothing's working, there's still particles that won't go into solution.

I need the samples for Western blotting and still need to quantify my samples as well.

I was thinking of leaving the samples at room temperature (they're on ice right now), maybe that would help?

Any suggestions would be appreciated.

Thanx,

RS

often scaffolding proteins are difficult to resolve after precipitation; as suggested most are to dissolve in Laemmli sample buffer; in this case, quantification is possible after gel-run with f.i. CBB staining; the disadvantage is the second run for Wb;

another possibility is to trying to dissolve pellets in a very low amount of DMSO or DMF;

sometimes recommended sonfication protocools may be harmful to proteins

Have you ever tried to laod DMSO on SDS gels? what would be the maximal concentration?
(I had a bad experience with methanol samples. I knwew it would not work, but I wanted to check in any away, so I diluted twice the methanol vehicle in loading buffer : surprisingly it was fine ! so I did the same with the sample, but I lost the few drops I started to load

. So now I'm a little more reluctant trying strange things !)

Protein pellet won't resuspend in buffer following acetone precipitation - (Feb/08/2007 )