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step-wise dialysis? - (Oct/15/2009 )

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Hi

I need the protocol of stepwise dialysis,in detail, and what is the ratio between the sample and the buffer in this type of dialysis?

-bluebird-

Step-wise is as it suggests, a step-wise reduction in the concentration of the component you wish to remove. Why do you want to do it? You are much more likely to get a quick response if you give us more background than less...

-swanny-

swanny on Oct 18 2009, 07:29 PM said:

Step-wise is as it suggests, a step-wise reduction in the concentration of the component you wish to remove. Why do you want to do it? You are much more likely to get a quick response if you give us more background than less...


I need to do it after extracting the protein from inclusion bodies, and now my protein is solublized in 8M urea.
the problem is I have the principle of the protocol but I don't know how to do it correctly.
this is the protocol:


((the pellet was resuspended in 5 ml 8 M urea in PBS, centrifuged as before and the supernatant retained. The urea-solubilized protein was diluted to 02 mg}ml with 8 Murea in PBS andthe urea removed by stepwise dialysis against 20 vols buffer A <25 mM TrisHCl (pH 72), 10% (v}v) glycerol, 50 mM NaCl, 1 mM DTT, 01% CHAPS> containing 4 M, 2 M, 1 M urea, then three buffer changes without urea. Each dialysis step was for a minimum of 2 h. The sample was clarified by centrifugation at 70000 g for 30 min at 4 C and dialysed
against buffer A containing 50% glycerol for 24 h and stored at20 C.)))

-bluebird-

That detail's great. OK, 20 vol buffer is 20x your sample volume. make up the first buffer with 4M urea and dialyse in that for > 2 hr. Then transfer the dialysis tube into the second buffer, made up with 2 M urea and so forth. The best way to do this is to make up the buffer without urea as a 2x solution (50 mM Tris, 20% glycerol, etc). Then add an equal volume of 8M urea to make the first (4M) buffer. Add 1/4 volume urea and 1/4 volume water to 1/2 volume of the buffer stock to make the 2M urea buffer, etc etc etc. But be warned that the protein might start to crash out because of unfavourable interactions as the urea concentration drops.

An alternate way to remove the urea is to have a large volume of the buffer stirring in a beaker. Add the denatured protein drop-wise into the buffer. This will rapidly dilute the urea away while reducing the risk of protein: protein interactions that might lead to aggregation.

If you still have problems, go to http://refold.med.monash.edu.au/ . It's a website dedicated to refolding denatured proteins, so you're pretty sure to find something of use.

Good luck!

-swanny-

swanny on Oct 19 2009, 05:21 PM said:

That detail's great. OK, 20 vol buffer is 20x your sample volume. make up the first buffer with 4M urea and dialyse in that for > 2 hr. Then transfer the dialysis tube into the second buffer, made up with 2 M urea and so forth. The best way to do this is to make up the buffer without urea as a 2x solution (50 mM Tris, 20% glycerol, etc). Then add an equal volume of 8M urea to make the first (4M) buffer. Add 1/4 volume urea and 1/4 volume water to 1/2 volume of the buffer stock to make the 2M urea buffer, etc etc etc. But be warned that the protein might start to crash out because of unfavourable interactions as the urea concentration drops.

An alternate way to remove the urea is to have a large volume of the buffer stirring in a beaker. Add the denatured protein drop-wise into the buffer. This will rapidly dilute the urea away while reducing the risk of protein: protein interactions that might lead to aggregation.

If you still have problems, go to http://refold.med.monash.edu.au/ . It's a website dedicated to refolding denatured proteins, so you're pretty sure to find something of use.

Good luck!


WOW description more than I expect, you described everything better than my supervisor. Thank you very much Swanny, :lol: :D :) :) have a nice day.

-bluebird-

just another question could you please explain the following:
((An alternate way to remove the urea is to have a large volume of the buffer stirring in a beaker. Add the denatured protein drop-wise into the buffer.))
also Can I store solublized protein in 8M Urea untilm the process in -80?

-bluebird-

There shouldn't be a problem. After al, the protein is unfolded already (the usual issue when freezing proteins), and the urea will have also denatured any proteases that might be hanging around.

-swanny-

Hi Swanny,

you didn't answer my question, how can Iadd the denatured protein drop-wise into the buffer, practically I don't have any Idea about this type of diialysis? <_<


2_also can I run SDS at this stage for solublized protein or I have to refold it first?

-bluebird-

bluebird on Oct 22 2009, 03:11 AM said:

2_also can I run SDS at this stage for solublized protein or I have to refold it first?

yes, you can run it. the sds will unfold the protein so no need to first refold.

-mdfenko-

mdfenko on Oct 22 2009, 10:06 AM said:

bluebird on Oct 22 2009, 03:11 AM said:

2_also can I run SDS at this stage for solublized protein or I have to refold it first?

yes, you can run it. the sds will unfold the protein so no need to first refold.



even if the protein soluble in 8M urea? do you think the SDS will give a good result???

-bluebird-
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