Can anyone tell how do I concentrate the protein in the dialysis bag by using PEG?The molecular weight of my protein is 20 kD the dialysis cut of is 14 kD.
First, I suggest you use a lower molecular weight cutoff, for 2 reasons: (1)The MWCO is a bit close to the MW of your protein and you don't want any protein to get through. (2) You want to minimize the amount of PEG that gets in. Try 2,000 or 3,000 MWCO dialysis tubing.
You make a concentrated suspension of high molecular weight PEG and put the dialysis tubing containing your sample in the suspension. The water is gradually drawn out into the suspension, concentrating the sample.
Another way to do this is to cover the dialysis tubing with dry carboxymethylcellulose. As the CMC stuck to the tubing becomes swollen with moisture from the sample, take it off and replace it with dry CMC.
Better still, use a centrifugal ultrafiltration unit or pressure cell with a 3,000 MWCO filter to concentrate your sample. It's faster.
Adam,
I surely use a low cut of membrane. will it be ok if i use 20Kd PEG suspension for dialysis bag with cur of 3kD.
@Adam B Shapiro
That's better than your previous plan. However, it is important to remember that 20 kDa is an average mass of the PEG in a preparation, and there may be some low molecular weight PEG in your protein sample at the end.
Adam,
No, i do not want any PEG molecule in my protein sample. Can u suggest me approximate molecular weight for PEG to be used in this experiment
@Adam B Shapiro
I'm not sure about this, but I suspect that any PEG solution will contain a small amount on low molecular weight monomer and oligomer that can get into your sample. Maybe someone else can comment on this.
I agree with Adam, but here are my 10 cents.
Use 20Kda PEG with narrow length dispersion range; then dialyze with a higher cut-off dialysis tubing to get rid of possible contaminating PEG. Dry PEG can be used also, not necessarily its concentrated solution. In any case, it helps to do the concentrating dialysis on a nutator to avoid an extreme local rise in protein concentration. Make sure to constantly monitor the process or you will face a risk of overdrying your sample.
Use 20Kda PEG with narrow length dispersion range; then dialyze with a higher cut-off dialysis tubing to get rid of possible contaminating PEG. Dry PEG can be used also, not necessarily its concentrated solution. In any case, it helps to do the concentrating dialysis on a nutator to avoid an extreme local rise in protein concentration. Make sure to constantly monitor the process or you will face a risk of overdrying your sample.
Good luck!
P.S. Centrifuge concentrators work best for most of the cases, but not if the protein is prone to aggregation.
P.P.S. Just noticed that the advice is very untimely . :)
In using PEG method, there is always some chance of PEG getting into the dialysis bag and contaminating the protein, as it is discussed above. Is it possible to remove that trace amount of PEG by dialyzing the concentrated sample against fresh PEG free buffer ?
That should work, but if the PEG concentration inside the bag is high, the osmotic pressure will result in the volume inside the bag increasing again.
can anyone cite some research articles where PEG was used to concentrate the sample in dialysis?
This is not a research article, but it is a useful description of some protein concentration techniques, including the PEG one.
https://bitesizebio.com/5813/the-in%E2%80%99s-and-out%E2%80%99s-of-protein-concentration-%E2%80%93-semi-permeable-membranes/
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