What is the mass of your protein..

~ 50 kDa

PS: Detergent exchange is not really feasable, because the functionality of the protein is lost in anything else than DDM or DM. (and Brij-35)

Patrick

Remove the imidazole by dialysis in a buffer without imidazole and DDM [ 100 x dialysis x 2]

then make a 30%-40% solution of PEG 20,000 with the same buffer, except lower salt say 50mM NaCl. dialyse your protein in this buffer...

this would concentrate your protein. PEG dialysis based concentration is old school but works fine.

Good luck

you can include DDM in buffers if it is important

Thanks for the advice!

Any advice on the dialysis membrane I should use? (I am afraid to enrich my sample with detergent that way and this will harm further experiments)

using the same detergent concentration may not enrich your DDM in sample...you can even go lower on the DDM in your detergent sample.. a 30kDa membrane should be good.

I meant lower DDM in your buffers

OK, thanks, I will give it a try :)

If you did not have luck, I would use a P10 or G25 in a spin column format. This keeps the dilution factor low. And I would change the DDM to a lower concentration in order to concentrate it with a spin column.

But, The best way to concentrate it is with ion exchange, if you can find conditions where it will stick.

We routinely used this procedure for the exact problem. Keep the DDM until you really have to remove it:

1. Dialyze the solution using standard dialysis tube.

2. Then use the dry "Concenrator Powder" here http://www.gbiosciences.com/PDF/Protocol/Concentrator_Powder.pdf

Quite easy with this powder. It concentrates fast, so watch the volume.

Good luck!

Funny, I figured out the same approach some months ago.

I exchange buffer now via PD10 columns (8 ml bed volume). I have to do that several times, due to a large IMAC sample volume.

Then I fill the best fractions into a 100 kDa cutoff dialysis membrane (Float-a-Lyzer) and cover it with Spectra Absorbent. I come down from 6 ml to 1 ml within 2 hours.

It SEEMS to work fine. Unfortunately, I do not have the techniques to determine the DDM concentration in the final sample volume.

But thanks for the help! :)

I am glad something worked for you.

Marcia

@ Patrick

Just one caveat with the Float-A-lyzer.. Every once in a while a cassette will leak !!!! Yes I learned the very hard way. conventional dialysis tubing works for large volumes.

Just a thought....

@Prem:

Thanks, I also learned the hard way that these types of Cellulose Ester membranes (unlike regenerated cellulose) cannot be used with dialysis clamps. It simply cuts the tube into half. (I have the 10 ml Float-A-Lyzer tubes with plugs)

Tie knots. Knots are way safer than any clips !! A tight knot will never come part.

My protein is of molecular weight 13.5kDa and there is loss of protein in centrifugal concentrators. Can i use sepahdex g25 to coat the dialysis membrane for concentration, will it work the same way as the spectra absorbent Please suggest if there are any other alternate means I use a 3.5k MWCO dialysis bag 

Never tried the Sephadex G25, I would give it a try. With Spectra Absorbent it works super nice.