I have a 24KD protein Eluted from Ni-NTA agarose by Elution buffer (NaH2Po4 50mM/ NaCl 300mM/ Immidazol 300mM/ pH= 8). Routinely, we dialysis this protein with dialysis bag (2 hr* 1liter dialysis buffer/ 3 time) then determine its concentration by bradford and perform activity assay. Then we concentrate it by millipore centrifugal filter (10 KD/ Regenerated Cellulose ) to crystallization. Since we finally use millipore centrifugal filter (10 KD) I think the dialysis bag is not only a time consuming task but also is redundant. Is it okay If i use millipore centrifugal filter (10 KD) instead of dialysis bag for buffer exchange, desalting and concentration?
Hi
Of course that you can use the ultrafiltration step for buffer exchange. No problem at all. I do not like dialysis since the protein has too much time to equilibrate in energy minima that could be potential traps for folding.
You can also use a PD-10 column or similar for buffer exchange and them the ultrafiltration for the final concentration step before crystallization
Best of luck.
Paco
Hi Zaiddodine, in order to remove the elution buffer by doing centrifugation filtration, you will have to add your dialysis buffer to the sample after concentrating it, spin, add buffer, spin, etc. until the sample is free of the elution buffer constituents.
You will have to decide if adding those steps will improve the protocol.
However, if you can use a desalting column, like Francisco suggested and then you can directly add your eluted protein sample to a spin filter and concentrate.
Dear Zaiddodine,
The serious of the steps described by Steingrimur will be the hell. Anyway the risk of agregation is huge. Please do not improve the protocol is working. Suggest you the trick, with additional centricone will remove agregates before crystalisation. The final sample spun down through the 50-100 MWCO centricone, take the bottom part will be free of agregates...
best - jan
Very interesting. I never notice that Amicon concentrator restrict imidazole less than 100mM. I use concentrators for buffer exchange (over 100mM imidazole), and never have problem (e.g. activity, crystallization). What problem could be? For people work on membrane protein, dialysis to large amount of detergent buffer is expensive. Of course, we could dilute it first, but it is time consuming.
So desalting column seems good choice.
Jarmila, thanks for the information! I also doubt about the detergent data in chemical compatibility table. I can't see a reason that you could use 5% sodium deoxycholate, but only 0.1% tween-20 or triton X-100. Deoxycholate is a harsher detergent than tween-20.
Yes you can used.Also you can easily wash your sample adding new buffer, and reequilibarting your sample in new buffer.But you have to consider lost of protein by adsortion to filter membrane. Calculate your yield. HNS
Dear Zaidoddine,
I never used a dialysis bag to get rid of imidazole after affinity purification of a HIS-tagged protein, but, as mentioned by others previously, first a desalting column to eliminate unwanted salts and imidazole, then a centrifugal ultrafiltration device with the appropriate NMWL to concentrate the protein.
With best regards,
Pierre-Henri
Dialysis and ultrafiltration are interchangeable and the choice usually depends on sample volume and available equipment and time. If you suppose that stability/activity of your protein may quickly decrease, ultrafiltration is better.
You can use Millipore centrifugal filters instead of dialysis bag for buffer exchange and desalting. However, it concentrate the sample which then causes too much and denser bands on the SDS-PAGE. On the other hand, they can be blocked during centrifugation which further increases the time of experiment. After the dialysis, you can use lyophilization to concentrate and also to make it powder. Good luck.
It's will work ... but all of them filter or dialysis bags will absorb a lot of your protein. Sometimes up to 50%.
I think it should be fine but is time consuming. You can also go for PD-10 desalting column.
Of course you can. I always use amiron 10 kD cutoff centrifugal filers to remove imidazole, urea, glycerol etc. from my protein solutions.
I was concerned about the ability of Amicon Ultra centrifugal filters to tolerate high imidazole concentration as well so I contacted Millipore. I was told that since the writing of the user manual, concentrations as high as 300 mM have been tested and that no adverse effects were observed with the filter.
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