DIY Protein Separation

[Mega [Andreas Stuermer]]

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

[Nathan McCorkle]

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 :)

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[Andreas Stuermer]

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.

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[Cathal Garvey]

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).

T: @onetruecathal, @IndieBBDNA
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[Andreas Stuermer]

Rough fractions would tottaly do it. At least at first. Thanks a lot for the idea!

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[Mega [Andreas Stuermer]]

Did a quick google research on it, no protocols found so far. How can I immagine dialysis step?

[bioscisam]

You could try something like His tagging where you add a few histidine residues to the end of your protein (i.e introduced in your expression vector) and then isolate on a nickel column such as HiTrap from GE Healthcare, there's probably cheaper suppliers of alternatives.. These come as mini syringe versions for small preps rather than Acta scale protein prep systems.

[Cathal Garvey]

I'd have suggested affinity chromatography, but it sounds like they're
on a "fishing expedition" and don't know precisely which proteins they
want, yet..

[Andreas Stuermer]

Yeah, the protein is unknown. We just know that one of the 10'000 protein of Coptis contains this enzyme that modified berberine. 

Maybe it would be able to bind berberine to a column and thus bind the broteins?!?

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[Andreas Stuermer]

*Maybe I would be able to bind berberine to a column and thus bind the proteins?!?

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[Mega [Andreas Stuermer]]

What about gel chromatography for proteins? It would separate them by size.

And seems relatively do-able? 

[Samantha Thompson]

Might be possible to separate them on/through sepharose if there's a
non-denaturing protocol. As for binding berberine to the column sounds
like an interesting approach although not sure of the chemical
reactions required, an organic chemist will definitely know more. Most
crosslinking kits seem to be for linking standard things like biotin
and streptavadin to things like oligos and antibodies, but am sure
there's a way to do it.
If resources/funds weren't a limiting factor I would try to assess the
activity of FPLC fractions, then LC-MS on those fractions to try to
identify protein sequences, then design degenerate primers to fish
their genes out and try to clone and express... I've never seen a DIY
mass spec though! One day maybe...
Otherwise sometimes you can get access to university central
facilities if you are associated with them or send to a CRO for
testing...
Basically these are the paths I'd want to one day see DIYBio work
around but currently I think that's the typical approach.

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[Josiah Zayner]

Gravity flow size exclusion should work fine. As someone else said use Sepharose or Sephadex. You can find it on eBay pretty cheap. If you have money to spare buying old (H)FPLC pumps is pretty cheap on eBay. I think there are people who do (H)FPLC on this list but people probably just don't have time or motivation to run samples for you, including me.

As Cathal said, An ammonium sulfate precipitation is a tried method that has been used for over 60? years. By increasing.decreasing the ammonium sulfate concentration you can precipitate out proteins and try the different fractions.

[Andreas Stuermer]

Gravity flow sepharose sounds like a way to go. Have you got a keyword how I can find a column where to put in the Sepharose? 

On Sun, Aug 3, 2014 at 8:39 PM, Josiah Zayner <josiah...@gmail.com> wrote:

Gravity flow size exclusion should work fine. As someone else said use Sepharose or Sephadex. You can find it on eBay pretty cheap. If you have money to spare buying old (H)FPLC pumps is pretty cheap on eBay. I think there are people who do (H)FPLC on this list but people probably just don't have time or motivation to run samples for you, including me.

As Cathal said, An ammonium sulfate precipitation is a tried method that has been used for over 60? years. By increasing.decreasing the ammonium sulfate concentration you can precipitate out proteins and try the different fractions.

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[Matt Lawes]

Chromatography column is the appropriate term. Typically glass or plastic. You may even find prepacked columns of sepharose / sephadex. Go to GE healthcare life sciences and look at sepharose / sephadex technical info to determine which resin (size exclusion) will work best for you.

>matt

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Andreas Stuermer <masters...@gmail.com> wrote:

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[Andreas Stuermer]

Thanks!! 

http://www.piercenet.com/product/disposable-plastic-columns

These also seem nice. Easy to use? 

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[Andreas Stuermer]

I ask ebay for "prepacked columns of sepharose"

Can u reuse them? 

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[Andreas Stuermer]

Probably by just running deionized water through them? 

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[Matt Lawes]

Yes sir ... those will work .. just pick right size / scale

Andreas Stuermer <masters...@gmail.com> wrote:

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[Matt Lawes]

Yes up until you have saturated the pores of the resin. These can be washed out to some extent, but will eventually be saturated. Unless you load heavily with unclarified supernatant, you will get many uses of each column.

>matt

Sent from my Verizon Wireless 4G LTE DROID

Andreas Stuermer <masters...@gmail.com> wrote:

Probably by just running deionized water through them? 

On Sun, Aug 3, 2014 at 9:00 PM, Andreas Stuermer <masters...@gmail.com> wrote:

I ask ebay for "prepacked columns of sepharose"

Can u reuse them? 

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[Cathal Garvey]

Can someone tell me what the difference is between sepharose and
agarose? Is sepharose agarose beads with known porosity for
chromatography, or is it just plain agarose, or chemically modified agarose?

I recall during research into nickel columns that sepharose was at least
based heavily on agarose with a few tiny changes, but I don't know
whether those changes were specific to the nickel immobilised version of
sepharose, or general to "sepharose", nor do I know in the latter case
what the changes accomplish..

On 03/08/14 19:39, Josiah Zayner wrote:
> Gravity flow size exclusion should work fine. As someone else said use Sepharose or Sephadex. You can find it on eBay pretty cheap. If you have money to spare buying old (H)FPLC pumps is pretty cheap on eBay. I think there are people who do (H)FPLC on this list but people probably just don't have time or motivation to run samples for you, including me.
>
> As Cathal said, An ammonium sulfate precipitation is a tried method that has been used for over 60? years. By increasing.decreasing the ammonium sulfate concentration you can precipitate out proteins and try the different fractions.
>

[Andreas Stuermer]

I don't know but (a good way to start a sentence :D ) I guess that pore width is very important. If pores are too small, proteins cannot diffuse into it and you don't get size separation (?)

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[Matt Lawes]

Sepharose is dextran conjugated to agarose beads. Pores absorb small molecules (salts etc) but proteins are excluded because too big (hence size exclusion chromatography) and run off the column. But if pore is too big you get nonspecific binding of the protein inside. Especially for peptides and small proteins. As I said before go look up sepharose at wikipedia and GE life sci website and you can quickly educate yourself sufficiently to decide what you need exactly.

>matt

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Andreas Stuermer <masters...@gmail.com> wrote:

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[Cathal Garvey]

Yea, I'm familiar with pore size and size exclusion chromatography, was
just wondering what made sepharose different from agarose. Thanks for
the answer! :)

On 03/08/14 20:36, Matt Lawes wrote:
> Sepharose is dextran conjugated to agarose beads. Pores absorb small molecules (salts etc) but proteins are excluded because too big (hence size exclusion chromatography) and run off the column. But if pore is too big you get nonspecific binding of the protein inside. Especially for peptides and small proteins. As I said before go look up sepharose at wikipedia and GE life sci website and you can quickly educate yourself sufficiently to decide what you need exactly.
>> matt
>
> Sent from my Verizon Wireless 4G LTE DROID
>
>
> Andreas Stuermer <masters...@gmail.com> wrote:
>
> I don't know but (a good way to start a sentence :D ) I guess that pore width is very important. If pores are too small, proteins cannot diffuse into it and you don't get size separation (?)
>
>
> On Sun, Aug 3, 2014 at 9:23 PM, Cathal Garvey <cathal...@cathalgarvey.me<mailto:cathal...@cathalgarvey.me>> wrote:
> Can someone tell me what the difference is between sepharose and
> agarose? Is sepharose agarose beads with known porosity for
> chromatography, or is it just plain agarose, or chemically modified agarose?
>
> I recall during research into nickel columns that sepharose was at least
> based heavily on agarose with a few tiny changes, but I don't know
> whether those changes were specific to the nickel immobilised version of
> sepharose, or general to "sepharose", nor do I know in the latter case
> what the changes accomplish..
>
> On 03/08/14 19:39, Josiah Zayner wrote:
>> Gravity flow size exclusion should work fine. As someone else said use Sepharose or Sephadex. You can find it on eBay pretty cheap. If you have money to spare buying old (H)FPLC pumps is pretty cheap on eBay. I think there are people who do (H)FPLC on this list but people probably just don't have time or motivation to run samples for you, including me.
>>
>> As Cathal said, An ammonium sulfate precipitation is a tried method that has been used for over 60? years. By increasing.decreasing the ammonium sulfate concentration you can precipitate out proteins and try the different fractions.
>>
>
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>
>
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