biotinylated proteins purification from free biotin - dialysis vs gel filtration or what else (Jul/19/2006 )

Hello,

I biotinylate a bacteria extract, and I would like to get rid of the free biotin, to be able to capture biotinylated proteins on streptavidin-agarose beads.

I already tried with a mini dialysis unit, but it seems it doesn't work well.
it's the one from pierce. I send them an e-mail, but meanwhile I would like to know what YOU use.

Thanks in advance

Missele

It should work. Maybe it is just you have not dialysized long enough. Regular dialysis tubing would be better, because of larger surface area. You can also use PD-10 desalt coulmn from Pharmacia or Sigma. What final conc. of biotin derivative did you use? You may also check if th conc. exceedes the solubility of biotin, thus longer dialysis time may be need. Good luck.

Thanks genehunter.
My sample is 100 µL, then the surface of the membrane is fine. The concentration of biotin is 2 mM, i don't know if it's too much.
I checked the PD-10 columns, but it would dilute my sample quite a lot.
I' m waiting for Pierce answer.
Let's see what they will recommend me.

Pierce says I should increase the amount of bead when I capture the biotinylated proteins.
the problem is that I'm absolutelly able to capture biotinylated proteins from biotinylated bacteria, in the same conditions.
the only difference is that when I biotinylate bacteria extracts, I can't wash them. Then there is 50 mM tris and 2 mM biotin in addition.
Tris and biotin should be dialyzed.

I tried to dialyze bromophenol blue (brecause it's colored, it's a visual test, and it has quite the same molecular weight), but after two hours (while the equilibrium should be reached in 10 minutes to 2 hours), the bromophenol solution is still as blue as before.


I'm pissed off.

with a narrow dialysis tube (1/2 in??), 100 ul is still doable, particularly when you are running into prblem with that kind.

1/2 '.

Biotin is a weak acid. i wonder pH may play a role here.

It should be sufficient, providing that pH is neutral or slightly basic. Can you use buffer, tris-HCl, pH 7.8 instead of water?

hello,

an alternative to mini dialysis is to use the biorad P6 spin columns. you'll only be able to remove the biotin from about 100µl of protein extract per column, but the good news it that you'll be able to get rid of it in about 10 min w/ minimal sample loss, and they come in packs of 25 or 50. i'm pretty sure you can re-use them, but i personally haven't tried it. just be sure to block the column w/ about 75µl 10mg/ml BSA before you run your sample over it (some of the acrylamide resin in them may not have polymerized completely, and ends up cross linking your protein if you don't block it). don't worry...start to finish, this will take no more than 15 min for a de-salted extract. if you have more questions about this, email them to me at jonmike.reed@gmail.com.