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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
>>>
>>> --
>>> -- You received this message because you are subscribed to the Google
>> Groups
>>> DIYbio group. To post to this group, send email to
>> diybio@googlegroups.com.
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>>> diybio+unsubscribe@googlegroups.com. For more options, visit this group
>> at
>>> https://groups.google.com/d/forum/diybio?hl=en
>>> Learn more at www.diybio.org
>>> ---
>>> You received this message because you are subscribed to the Google Groups
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>>> Visit this group at http://groups.google.com/group/diybio.
>>> To view this discussion on the web visit
>>>
>> https://groups.google.com/d/msgid/diybio/368c5577-7d1e-468b-8ded-6e2d7ffa5a12%40googlegroups.com
>> .
>>> For more options, visit https://groups.google.com/d/optout.
>>
>>
>>
>> --
>> -Nathan
>>
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>> .
>> For more options, visit https://groups.google.com/d/optout.
>>
>
>
>
--
T: @onetruecathal, @IndieBBDNA
P: +353876363185
W: http://indiebiotech.com
Re: [DIYbio] DIY Protein Separation
Read User's Comments(0)
Re: [DIYbio] DIY Protein Separation
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.
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 :)
> You received this message because you are subscribed to the Google Groups
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
>
> --
> -- You received this message because you are subscribed to the Google Groups
> DIYbio group. To post to this group, send email to diybio@googlegroups.com.
> To unsubscribe from this group, send email to
> diybio+unsubscribe@googlegroups.com. For more options, visit this group at
> https://groups.google.com/d/forum/diybio?hl=en
> Learn more at www.diybio.org
> ---
> "DIYbio" group.
> To unsubscribe from this group and stop receiving emails from it, send an
> email to diybio+unsubscribe@googlegroups.com.--
> To post to this group, send email to diybio@googlegroups.com.
> Visit this group at http://groups.google.com/group/diybio.
> To view this discussion on the web visit
> https://groups.google.com/d/msgid/diybio/368c5577-7d1e-468b-8ded-6e2d7ffa5a12%40googlegroups.com.
> For more options, visit https://groups.google.com/d/optout.
-Nathan
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/CA%2B82U9%2BchZ3c79QH29EdzokwLMHs1NmRYqJcvYH_iLxnyEwBRQ%40mail.gmail.com.
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