Re: [DIYbio] DIY Protein Separation

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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 <masterstorm123@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 <cathalgarvey@cathalgarvey.me<mailto:cathalgarvey@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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