I am trying to purify my enzyme from plant source. I used 0-80% ammonium sulphate, can I use gel filteration using sephadex G-50 resin to desalt my sample instead of dialysis. As dialysis leads to very much dilutiion of my sample. Thanks.
You can use Sephadex G-50 or another gel filtration resin to desalt the sample, but the result will also be sample dilution. To avoid that, you can use gel filtration spin columns.
As rightly quoted by Dr. Shapiro, you can use Sephadex G-25 (Commonly used for desalting) and even Sephadex G-50. An alternative could be use of 10KD, 30KD Centrifugal Membrane Devices (Offered by Millipore and Pall) which can not only desalt your protein but will also concentrate them.
Well! In order to use/select the Gel-filtration spin/columns cut-off, you should know the molecular weight of your enzyme. If it is not mentioned in the literature or reported to have a wide range of molecular weights in different sources, then you have to perform the Native/SDS-PAGE analysis to know the molecular weight (you need to do the activity staining on Native-PAGE of the same gel part, to know which band is of your enzyme of interest. And if this is not possible then you have to depend on the literature for the molecular weight). And for this you need to have a concentrated enzyme sample.
My suggestion is, after salt removal using the dialysis membrane, just put this same dialysis bag (containing your enzyme sample) in a sugar (solid) or in Glycerol which will withdraw the buffer out of the dialysis membrane and your protein will ultimately get concentrated.
I really think it depends on your sample volume. If you have low volume (1-10 mL?) then G-25/50 spin columns, or a short stack conventional column may be the quickest and easiest. They work very well, and a short stack column can be easily be calculated using 1 ml fractions, or less, to exclude the salt fractions. However, there will be significant dilution with short columns, much less with spin columns. I have used Pierce "Slyde-A-Lyzer" pancake-type dialysis cartridges for 1 mL +/- volumes. Larger volumes require multiple spin columns or a short stack gel filtration. Low molecular weight pore tubular dialysis membranes are fine for about >5-10 mL, and the dilution is minimal, certainly less than a G-25/50 stack elution (but I guess not in your experience?), but slightly greater than a spin column. In most instances, for larger volumes, the tried and true LMW cut-off dialysis membrane is the way to go.
Be careful add ur protein sample step by step diluted with buffer of interest , in case you tried to desalt all the protein at once using the desalting Centrifugal devices, it may harm the membrane but yes you can do both buffer exchange and desalting
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes