SEPHADEX (Pharmacia)?

[noam]

Hi, I was wondering if anyone has used SEPHADEX.
I'm trying to desalt proteins with mass of 8000-15000.
I've tried the G-10, which should have given me a 12000 protein in the void, the problem was that the salts are coming to close to it (very little salt separation?), I've increased the amount of G-10, but no luck.
I thought that going to G-25 would improve my separation but nothing.

Maybe it's the protocol I'm using?
I've tried mixing it with water from the box, tried letting it swell quietly or with heating- and nothing.

I've asked around and very few people seem to work with it, so if someone have used it and could help me out I would really appreciate it.

Thank you, noam

[sabine]

Hey noam!
It's a while ago since we used Sephadex...
As far as I remember we used 25% ethanol as swelling and packing buffer. We (gently!) mixed the dry medium with buffer, let it swell over night and packed the next morning.
You may find updated and extensive information on the homepage of the manufacturer - Pharmacia became Amersham Biosciences and is by now part of GE Healthcare: http://www.amershambiosciences.com

I hope this helps a bit!
Sabine

[noam]

Sabine hi, thanks for the reply.
Do you know if there is a reason not to let it swell in 100% H2O, I thought that the use of ethanol was mainly anti bacterial?
The protocols I've seen mentioned "swelling in buffer", but not the making of the buffer.
I was originally thinking of using 100% H2O as my running buffer, so I've tried swelling in same buffer, maybe swelling in water impairs mechanic behavior?

[sabine]

Hello noam,

we generally try to use ethanol for column packing - as long as its compatible with the separation medium. That is mainly due to reproducibility, simplicity... And to prevent microbial growth, of course (I work in the pharmaceutical production area. Microbial growth is an important issue here )

At the moment I can't think of reasons not to use water. Maybe you could contact your local GE HC/Amersham representative?

Or your problem is not the packing buffer but something else in your procedures (packing itself, column dimensions, sample volume...) - can you give any more information?

[noam]

Thank you, very much Sabine

I'm afraid that self packing these things is not so common cause GE is pushing the new and improved prepacked product, being in university means small bought...

I've tried various dimensions, do you think that I could get a rough estimate of Sample volume by using under 30% of void volume (i kept the volume under it)?
During swelling what kind of mixing would you use?
Would you pack it under atmospheric pressure?
after swelling, would you take some sort of separation step i.e. getting rid of "broken beeds"?
How stable is it in dry form (straight from the box, Since some of the phases I've used have old Pharmacia logo on the box)?

[sabine]

Packing a gel filtration column can be challenging... You should also check the handbooks on the GE page (especially "Gel filtration -
Principles and Methods" of course) - these are really informative!

I think, your sample volume should be OK.
We usually prepare slurry directly in the column, but this is just applicable for larger colums (10 cm+). So just use another suitable container.
To prepare the slurry we add powder to the buffer and stir gently - in lab scale don't use a magnetic stirrer, but a glass rod for example! Else the bead might be broken.
When the slurry is homogenous we let it just sit and swell until next morning (no further stirring). Then we remove part of the supernatant (slurry settles a bit over night and in the supernatant swim the fines - broken beads - if any), maybe resuspend again until homogenous and pack the column by flow packing. (In your case: carefully pour prepared slurry into column. Try not introduce air bubbles! This is quite important with gel filtration).

What you could try after packing is a performance test with acetone as sample and water as mobile phase! This way you can easily check the packing without wasting product... This is also described in that handbook by the way...

concerning stability: every box should state an expiry date?! But I'd say that the dry powder might be stable longer than that date. It's just what the manufacturer guarantees!

[mbreslav]

Size exclusion chromatography has relatively low loading capacity tolerance. I would try to a)decrease loading; and b)decrease sample volume. Injection should be made very carefully by distributing sample solution in an even plug all across the surface on the top of the column. Good luck.

[noam]

Thank you all very much,
I'm going to try all the above, I had a tragedy in my family, so I'd take a couple of days off though…

Just last question, what do you do to minimize disturbance to upper phase when applying sample on open (atmospheric) column?

[mbreslav]

If your column is opened to air, then you need to make sure that it has a minimum amount of the residual mobile phase over the top of the stationary phase without drying the Sephadex. I used to load sample solution with a pipette very carefully without disturbing the layer of the phase in a narrow plug of liquid. Then I would pour some mobile phase in a beaker and very carefully and slow let sample solution suck into the phase by allowing the gravity flow from the column. As soon as your sample solution is almost adsorbed, you need to start adding mobile phase with a pipette so to not let Sephadex dry. Your goal is to create a narrow plug of the sample solution inside the column without drying the phase. When sample solution is adsorbed, then you can start your separation safely and slow. Always make sure that you don't allow air inside the phase.
Your mobile should be designed to prevent any affinity interactions between your solute and Sephadex leaving only size exclusion. If your compound interacts with the stationaty phase, you need to have a stronger mobile phase (add acetic acid, or alcohol), or switch to other types of Sephadex (LH-20 can tolerate organic solvents, like DMF).

[sabine]

Sorry, I assumed you were using an automated system! I have never used open columns...
I think mbreslav is also right about your mobile phase. Is it possible for you to add at least some salt or even buffer substance? I don't know what you will exactly need afterwards or what your sample is, so it's not easy to suggest anything. But I would maybe try 10 mM of a suitable salt or buffer...

And I just remebered that it is usually better to use a long, narrow column than a short, wide one for gel filtration, although I think this shouldn't be too critical with group separation and sephadex.