Hello there,
Sorry for so many questions in one go. I have purified four His tag proteins using His60 gravity flow columns from Clontech. I had good yield of the proteins after and dialysis (dialysis buffer 50mM Tris, 100 mM NaCl, 1mM EDTA+10uM Pefabloc SC) but the volume was quite large (12-25 ml). So I decided to use Vivaspin 20 columns with an appropriate MWCO to concentrate the proteins. Out of four proteins, I managed to concentrate two proteins satisfactorily, although I lost more then 50% of the proteins. For one of the remaining proteins, I get precipitation when I concentrate it using Vivaspin 20 column, while for another I just lost more then 80% protein. Also it seems that something was blocking the membrane of the Vivaspin column as it took a long time to reduce the volume of the last protein. I wonder, if it was my protein of interest sticking to the membrane? Thanks for suggestions in advance.
Hi,
Most likely the protein was precipitating during the spin and the precipitate was blocking the pores non-specifically. In terms of concentration after dialysis we always used dialysis against PEG-20,000. You don't need to take the sample out of the bag, just lay it on a bed of high MW PEG, and cover it with the same. The osmotic pressure will remove much of the water and the high-MW PEG will be unable to enter the dialysis tubing. Should concentrate at least 10-fold or higher. Do it in the fridge or cold-room and your protein should be fine.
Cheers,
Dave
Hi,
Most likely the protein was precipitating during the spin and the precipitate was blocking the pores non-specifically. In terms of concentration after dialysis we always used dialysis against PEG-20,000. You don't need to take the sample out of the bag, just lay it on a bed of high MW PEG, and cover it with the same. The osmotic pressure will remove much of the water and the high-MW PEG will be unable to enter the dialysis tubing. Should concentrate at least 10-fold or higher. Do it in the fridge or cold-room and your protein should be fine.
Cheers,
Dave
Thanks for suggestions. @ Paul: What do you mean by the protein will crash out? Will it denature or what?
Thanks guys.. @ Matteo.. I tried speed ranging from 2500 g to 4000 g, same outcome. Protein precipitated.
@ Gianne: I was pipetting up and down for 15-20 times after every 10 min of centrifuge. The calculated pI of my protein is around 6 and I used buffer with pH 7.5. I think it should be ok. I think, I will give PEG a go and let see what happens..
Hi!
Sometimes happens that your protein precipitates or aggregates during concentration with vivaspin. Most of the previous comments should help you, but I think that the most important thing for you is to try to reduce the rpm and to try absolutely at temperature lower than 20 °C. I suggest 4° C...You should increase your protein yield. Good luck!
Dear Sandeep,
in our experience, for such large volumes and "gentle" proteins, the Diaflo stirred cells (http://www.merckmillipore.com/PT/en/product/Stirred-Cells,MM_NF-C3259) work best.
I also agree with the colleagues that suggested the 4degrees C centrifugation. We always do our concentration in these Diaflo apparatuses at 4degrees C.
Hope this helps, cheers,
Jose
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