Paul Cullen wrote:
> 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
Hello Paul,
I don't know if this is of any help, but what I usually do is the following:
For the soluble proteins, before acetone precipitation I assay protein content
to have an idea of the amount of resuspension buffer I use. After precipitation
and centrifugation, I discard the supernatant and let the tubes to dry in the
fume hood for few min, just until I can barely smell the acetone, small amounts
of it have not been a problem for my 2-D gels. Then I add 40 ul, per mg of
protein, of 10% SDS, 1% DTT in water and I vortex vigorously to solubilize the
pellet. Some of my samples don't get solubilized and I have to boil them for 3-5
min. Here you can freeze the samples, or add 210 ul (per mg of protein) of the
sample buffer, may be you can get it from the proteomics place if they want
special sample buffer to be used, I don't see why they can't provide you with
some of it. Although SDS is not recommended for IEF, it is well diluted (5-7
times) in the loading buffer (the one I use contains 9 M Urea, 2% CHAPS, 1% DTT,
and 0.8% ampholites). Some time I forgot some samples, after acetone
precipitation, drying in the hood and they were dry such I couldn't resuspend
them at all, so don't dry them too long.
I could think that this also would work for your triton samples. I have
tried to do membrane proteins and before I used a lot of different detergents
for solubilization, but I ran into lots of troubles, then what I finally did was
to not to use any detergents prior the sample buffer for 2D gels, I just
pelleted the membranes by centrifuging at 30 000Xg for 20-30 min, remove the
supernatant and either freeze the membrane pellet or resuspend it to the sample
buffer (same as before). I usually get nice looking gels.
I have bad experience with PBS, it seems that it is not compatible with some
of the conditions for the isofocusing, can't remember exactly how. In any event
I always try to avoid PBS.
Hope this helps.
Cheers,
Arturo
_______________________________________________________________
Arturo Garcia-Horsman, Ph.D.
Research Specialist
Department of Pharmacology and Toxicology
University of Kuopio
Canthia, Harjulantie 1 A, 1. krs.
70210 Kuopio, Finland
Tel: +358-017-162422
Fax: +358-017-162424
E-mail: Arturo.Garcia-Horsman at uku.fi
