Hi
We use acetone precipitation for hemoglobin adducts to get rid of the
chemicals used for coupling. Normally after drying, these pellets dissolve
very well in saline (not in PBS!) but for some reason, one particular
chemical ?denatures? the protein that much that we cannot dissolve it any
more. We used ultrasonification to dissolve at least part of it.
One question, does the acetone interfere with what you want to do
afterwards? If not, you could try to dilute the acetone with saline until
the pellet dissolves (works for hemoglobin) or even try to get rid of the
acetone by, e.g., dialysis.
Hope it helps,
Marty
--
Marty Wulferink
Div. of Immunology
Med. Inst. for Environmental Hygiene
Duesseldorf, Germany
Paul Cullen <Paul.Cullen at med.monash.edu.au> schrieb in im Newsbeitrag:
3B091A8A.7903190B at med.monash.edu.au...
> Hi everyone,
>> I am acetone precipitating proteins from the aqueous and detergent
> phases of a Triton X114 extraction. The problem is that the pellets if
> allowed to dry become hard like rocks and insoluble in normal 1D sample
> buffer for protein gels. I want to store the precipitated proteins for
> several weeks before I go to a specialised proteomics facility. I am
> worried if I freeze dry these pellets that they won't be of any use when
> i get to the proteomics facility. These are the ideas I have come up with:
>> 1) Leave the proteins in acetone solution.
> - I've herd this can also make the pellet impossible to resuspend.
>> 2) Try to resuspend the samples in PBS and then freeze dry or store at -70
> - I can't resuspend them in sample buffer because the proteomics place
> where i'm going has proprietary detergent solutions which they will
> resuspend them in.
> - I can't imagine the detergent phase proteins would go into a PBS
solution.
>> 3) Don't resuspend them in PBS, just layer it over the top of the
> pellets and store at -70.
>> Please help or give any suggestions that you can.
>> Thanks,
>> Paul
>> --
> Paul Cullen
> Bacterial Pathogenesis Research Group
> Monash University
> Australia
