inclusion body had dissolved in 1M urea... - do I have to dialyse it? (Apr/18/2006 )
By mistake i have added 1M urea instead of 8M urea and my inclusion body half dissolved... ...ill try again with 8M urea but i want to use the sample i have prepared since i guess maybe its enough for my experiment....but now im thinking against what should I dialyze it?? ....i mean should I remove urea at all?? like i dialyze against a solution with no urea for couple of hours?? thank you again!
The fact that your protein re-solubilised with 1M Urea is great. Your mistake has worked out well for you as many times the next experiments you use the purified protein in are inhibited by urea.
In addition, the lower amount required to solubilise the protein may mean that there's a less likely chance that it will precipitate out when you dialyse the urea out of solution.
You need to decide on whether the urea conc will inhibit future experiments ie
- enzyme activities if you are cleaving the protein,
- if the protein is for antibody production etc.
- or for function assays of the protein itself (if this is the case you may be in trouble as re-solubisation does not always mean correct refolding).
As the urea conc is relatively low in terms of getting the protein to resolubilise I would give dialysis a go as it may not precipitate out, or you could at least decrease the urea conc to mM.
If you let us know what you need the protein for and your plans for future experiments we may be able to guide you better.
thank you very much! I need to cleave my protein with a protease. So you suggest I dialyse against 0mM urea? and hope that my protein doesnt precipitate right?
But...If you dont want to put all your eggs in one basket then try to find out the if the protease you are using is inhibited by urea. One enzyme I was using was ok if I decreased the concentration to 250mM urea. This allows some stability to remain for your protein yet still allows you to continue without inhibition.
As you are unsure whether it will precipitate you could do two lots of dialysis; one straight into buffer with no urea and one a step-wise decrease of urea conc.
but i always thought that im removing urea in order for my protein to refold... ...i mean if it not in the proper structure protease might not cleave it...in another words how can I be sure that my protein is in its native structure if i didnt remove urea...??? or what you mean is that 1 M urea is not that concentrated to denature my protein in the first place? .... ....thanx a lot again!
My understanding is that urea is strong denaturing agent that aids in the re-solubilsation of a protein from an inclusion body. This does not necessarily mean that your protein has correctly refolded upon the addition of urea.... especially if the protein "falls" out of solution..ie precipitates once urea is removed.
However, if you do not need the protein to be active then dont worry as the enzyme should still recognise the cut site for cleavage. Most cleavage sites are quite short and structure of the protein will not matter. The inhibitory factor is more likely to be the presence of too much urea.
If you do require an active protein you will need:
- see if the resolubilised protein has refolded correctly and has activity once the urea is removed,
If it hasn't you may need to:
- refold the protein using a kit you can buy (however I have never done this and have heard it can be very difficult)
- or try to express your protein in a soluble form by altering temperatures, induction time and IPTG concentrations.
To help you understand what urea does go to the following website (http://www.answers.com)and type in
"Urea is a powerful protein denaturant. This property can be exploited to increase the solubility of some proteins."
"A denatured protein is one which has lost its functional conformation. Once denatured, a protein loses most, if not all of its biological activity."
"In some cases, denaturation is reversible, and proteins may refold. In many other cases, however, denaturation is irreversible."
thank you so much for the explanations! i have done experiment with 8M urea again and ive got much better results (pure and abundant) but when i tried to dialize it against urea i did 6M and it was fine, then 4M and my protein precipitated... ....now im worried myabe thats because i was using DW and not DDW or MQ.....do you think it can effect the dialysis? or maybe its just precipitated as I removed urea?? my professor suggested i add beads (GST) to the protein before it precipitates ....but 4M urea is too much ?!?! ...what do you think I should do???
i wud worry about the dialysis conditions , urea concentrations and protein condition. DW, DDW n MQ water will not have any drastic differences. just to get rid off all these kind of small problems, try reduce variation in ur experiments as much as u can.
for most proteins the differences between dw, ddw and mqw would not make a difference, but there are some proteins that are very sensitive to contaminants that distillation doesn't remove. we worked with one such protein. we had dw taps which we ran through mixed bed ion exchange to polish the water. our protein was very unstable in this water. then we added a activated charcoal cartridge to the system and our protein stabilized. apparently, due to the nature of continuous distillation (as opposed to fractional distillation), organics were contaminating our water. these organics destabilized our protein.
so, sometimes water quality does make a difference. it won't hurt to try the mqw in your solutions.
may be you should try to dialyze the sample in 1M urea against the buffer under which your protein refolds (I use a phosphate, NaCl, for refolding from inclusion bodies in Gdn 6M).