dialysis questions - (Aug/03/2007 )
HI!
I have a couple of questions concerning dialysis. I already tried gel filtration instead of doing dialysis and it worked fine but it is more expensive and I cannot process a big amount of material... thus I switched to dialysis. My main aim is to remove nucleotides or other low-melucular weight contaminants from my lysate.
1) I'm doing it o/n and against 100mM Tris 0.1%SDS... To prevent protein degradation (and in my case to preserve phosphorylation) I'm performing it at 4°C... Shouldn't SDS precipitate?
2) As I'm quite worried about protein degradation or dephosphorylation, do you think would be good to increase the SDS concentration to 1%? Do you have any suggestions how to limit protein dephosphorylation or degradation?
Thx a lot!
Hi Sephadex!
What do you mean under degradation ( proteolytic degradation ( if so you should use appropriate protease inhibitors during dialsis) I don't think that SDS helpyou in this case ( if only you are afraid of solubility your protein during dialysis) I think it is not a good idea to increase % of SDS ( possibiltity of denaturation of protein will increase) . Direct answer about 0.1% SDS precipitation at 4C - Don,t worry about precipitation ( It will not precipitate) but worry about denaturation!
well, for protein degradation, there is PMSF which is active first 30'. Cocktails contains molecules which are longer active.
For dephosphorylation, use NaF or Nadeoxycholate.
Don't increase the %SDS, i think it will more denature your proteins than preserve from degradation.
Hello! Thx a lot for your reply!
Yes! I have some concern about the proteolytic degradation of my proteins as they are in the dialysis bag for the whole night.
Concerning protease (and phosphatase) inhibitors I cannot use them as their MW is smaller than the membrane cut-off (10-14kDa). I'm using pepstatin, benzamidine, PMSF, leupeptin,... as protease inhibitors and NaF, NaN3, pNpp, NaPP,... as phosphatase inhibitors... So if I want to use them I should put them in the dialysis beaker as well and I don't know if my prof. will be so happy to throw away the whole precious protease inhibotors cocktail for one experiment.
Therefore I just thought to increase a bit the SDS concentration (I read in a paper they were dialyzing against 1% SDS) to be sure to really inactivate the proteases and phosphatases.
By the way, is someone aware about a good phosphoprotein standard to spike into a lysate to determine wheather and to which extent in the subsequent steps dephosphorylation have occurred? I'm extracting proteins from yeast and after precipitation I'm digesting them with trypsin...
bye! 
Concerning 1% SDS - the authors did it with the same proteins? If so why not to do so?
But why you mention about high price of GF method? Sephadex G 25 is not expensive To get rid of low molecular impurities you may apply protein sample up to 20% of column volume. Have you possibilty to concentrate your extract ?
Yes! I have some concern about the proteolytic degradation of my proteins as they are in the dialysis bag for the whole night.
Concerning protease (and phosphatase) inhibitors I cannot use them as their MW is smaller than the membrane cut-off (10-14kDa). I'm using pepstatin, benzamidine, PMSF, leupeptin,... as protease inhibitors and NaF, NaN3, pNpp, NaPP,... as phosphatase inhibitors... So if I want to use them I should put them in the dialysis beaker as well and I don't know if my prof. will be so happy to throw away the whole precious protease inhibotors cocktail for one experiment.
Therefore I just thought to increase a bit the SDS concentration (I read in a paper they were dialyzing against 1% SDS) to be sure to really inactivate the proteases and phosphatases.
By the way, is someone aware about a good phosphoprotein standard to spike into a lysate to determine wheather and to which extent in the subsequent steps dephosphorylation have occurred? I'm extracting proteins from yeast and after precipitation I'm digesting them with trypsin...
bye!
addition of SDS during dialysis sounds strange as it will denature most proteins but less proteases which will become really active as they often target denatured proteins!! moreover, as a long-term effect, SDS will precipitate at 4°C
phospho standard: depends on how cells has been stimulated before lysis; good targets are membrane receptors, f.i. if stimulated with EGF you may follow the activation (cisphosphorylation) by appropriate anti-phoshp EGF-R Ab´s
4 degrees should be cold enough to inactivate the majority of proteases, I wouldn't worry about it unless you know for sure you have a degradation problem!