Re: [DIYbio] DIY Protein Separation

How precisely do you need them separated? Would rough fractions do, say
10-20 fractions based on solubility in ammonium acetate solutions?
Ammonium acetate is a very gentle way to separate proteins I'm told,
it's generally easy to re-solvate folded protein after separation for
activity assays (although you'll need to do some dialysis).

On 01/08/14 09:37, Andreas Stuermer wrote:
> SDS would not be nice because it'd denature the protein.
>
> Preperative sounds nice, as I want to see enzyme activity afterwards.
>
> HPLC we have at uni.
>
>
> On Fri, Aug 1, 2014 at 1:27 AM, Nathan McCorkle <nmz787@gmail.com> wrote:
>
>> wouldn't you use protein SDS gel electrophoresis to separate the
>> protein? or is that too analytical and you want preparative (i.e. you
>> need a lot of your pure protein)?
>>
>> I guess how much protein do you need? I seem to recall Josiah posting
>> on biocurious that he was cleaning up a F/H-PLC to try and get
>> working... so I presumed he has some experience with them. I've some
>> HPLC and GC once in a class, and according to wikipedia ion exchange
>> chromatography is also considered FPLC sometimes if used for protein,
>> so when I used the Carolina kit for GFP I guess I was doing FPLC :)
>>
>> These terms are all a bit overlapping, kinda like the act of walking,
>> whether you have sandals or boots on. A lot of similarity, but
>> differences around the edges.
>>
>> normal phase vs reverse phase (is the buffer (mobile phase) polar or
>> the resin (solid phase) polar?). You can also have different phases
>> than solid and liquid. You could say that your lungs filter oxygen and
>> repel CO2, that would probably be something like gas to liquid to
>> solid... but that might be stretching it for an /easy/ example :)
>>
>>
>> On Thu, Jul 31, 2014 at 6:09 AM, Mega [Andreas Stuermer]
>> <masterstorm123@gmail.com> wrote:
>>> Hi!
>>> Again to my question, there seem to be little guys dealing with FPLC
>> here.
>>>
>>> https://groups.google.com/forum/#!topic/diybio/24PZTzije98
>>>
>>> Is there a way to do this cheaply at my university, without buying an
>> FPLC
>>> machine?
>>>
>>> Thanks god those berberine derivatives are all fluorescent. So detecting
>>> will not be a problem.
>>>
>>> What we want to do is: preparing a cell protein extract. Separate those
>>> proteins. Add the fractions to berberine, let it incubate,  and do
>>> photometry see which turns it into the derivative we want.
>>>
>>> Best,
>>> Andreas
>>>
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>>
>>
>> --
>> -Nathan
>>
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>
>
>

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