changing dialysis buffer - (Nov/15/2006 )
i was wondering if anyone here who does protein purification changes dialysis buffer during dialysis? usually i leave the membrane in the buffer overnight and remove the protein in the morning.
When I was doing antibody purification, I did three changes of the same dialysis buffer.
I don't know about protein.
The purpose of doing three of more changes of dialysis buffer
1. reduce the the volume of dialysis buffer use, ie. use smaller volume
2. dilution of the unwanted salt and detergent in the protein solution to undetectable amount
I hope this may help.
Without changing the dialysis buffer, there will be residual salt in the solution. The best you can do is distribute the salts originally present in your sample uniformly within the volume of sample+buffer, effectively diluting the salts by the ratio of the buffer to sample volume. By changing the buffer, this dilution is squared, and a second change cubes the dilution factor.
I agree. I always change 3 times.
i use 2ml of protein extract in 500ml dialysis buffer.
do you think it's uncorrect? 
I used to know a dilution factor that my boss gave me for how much sample vs. dialysis buffer volume to use, but I forgot it. Now I just use 2-8ml sample in 2L buffer and change it 3x, but it is most likely way overkill. I probably could use a smaller volume, but I am not exactly sure what the rule is anymore. Does anyone know?
do you think it's uncorrect?
for most purposes your dialysis is fine. you get a 250x dilution of the salts.
for more sensitive procedures you may use the same total volume in parts and get a much higher effective dilution of salts, just make sure that each step is dialyzed to near equilibrium (~2 hr, i think, also dependent on surface area/volume of dialysis tube).
WAstate, isn't using 8L of buffer for 2-8 ml sample a little wasteful? or are you dialyzing against water (which won't matter re: waste unless you use bottled)? you can achieve the same effective dilution by dialyzing against 200 ml with 5 changes (6 volumes of 200 ml).
I definitely think it's wasteful, but that is how I was taught, and I just didn't know any differently.