Problem with protein dialysis - (Apr/04/2006 )
I have precipitated supernatant proteins with acetone and resuspended in 200ul water. I am de-salting them by dialysis for 1h in 1 liter water as dialysis "buffer", but I should be dialysing 3-4h hours according to my mentor, but my samples look like they are getting too diluted after half an hour and like they are going to spill over from the collodion dialysis bag!
Is this happening because I have the proteins resuspended in water which is also my dialysis buffer? So water readily goes in after the equilibrium is reached? After I resuspend in water, if I use PBS saline as a dialysis buffer, do you think this overspill wouldn't happen? How do I prevent that!?
By the way, I am only resuspending in water for now because we are just interested in seeing what proteins are found in the supernatant, I know I should be resuspending in PBS or some other buffer.
Any help much appreciated:)!
You have precipitated your proteins and then solubilized them again, but solubilized proteins are not visible for the eye and if the cut-off of the dialysis bag is lower than the kDA of your proteins, they never can get out of the bag. You want to get rid of the salt and I think that you see the salt coming out of the bag. If you have closed the bag properly, no water can come in, only salt will go out, so your proteins cannot be diluted more (or the cut-off of your bag is too high).
A couple of hours is even short, I dialyse at least over night.
A couple of hours is even short, I dialyse at least over night.
No, the cut-off is 10kD's which we don't really care about those small proteins, so that's ok. These collodion bags are the open kind, they have no closure, that is why I am seeing the volume rising above "threating" to bubbule out. I am not saying that my proteins are "escaping" from the bag, they are being diluted because all of the water coming in. I would like to prevent that. Any ideas, anybody?
okay, sorry.
Can you make a closure yourself, tie a knot in it (I don't know how they look)
if you have a speed vacc. or a lyophilizer you can concentrate your protein even if it gets diluted or you can even use a centricon to concentrate your protein of interest.
well also tell me what salts you trying to remove? media salts????
well also tell me what salts you trying to remove? media salts????
Yes, I will use centricon. And yes, it's the media salts I want to remove, the bacteria we are studying are marine bacteria that require a lot of salt.