Hi,
I am working on recombinant protein production and I want to know whether I can remove urea and triton-X100 with dialysis.
After the lysis of my cell pellet, I usually use 2M urea with triton-X100 buffers to wash protein pellets. (By the way, My protein is insoluble form.)
If anyone have experience about Urea and surfactant removal with dialysis, please share your experience with me. (or reference paper).
Thanks.
Urea is easily removed by dialysis. Triton X-100 is more difficult because it forms large micelles that don't pass through the tubing. I'm not sure whether the presence of urea makes Triton dialysis easier. If not, another common method to remove Triton is binding it with Bio-Beads. But if your protein is insoluble in urea + Triton, then you can remove them by centrifuging the protein and discarding the supernatant, and washing the pellet in buffer without them.
Do you have traces of triton left after cleaning the inclusion bodies, assuming you already do inclusion body harvests with a few water washes as Adam points out? The urea would likely be gone at that stage. Trying to dialyze away triton is difficult if you still have trace amounts left and essentially just water remaining with the inclusion bodies because the triton CMC is in tenths of millimloar with a large aggregate number in the 90 Kd range under those conditions. It would take a some time by dialysis unless you can get below the CMC. Since you have inclusion bodies then perhaps organics such as ethanol, methanol, and isopropanol or cyclodextrins would flush out any Triton adhering to the inclusion bodies and avoid having to remove resin beads mixed in with the inclusion bodies.
Hi ,
First Convert your insoluble protein to soluble form.
SDR Hyper D is well known for Detergent like Titron X 100 removal ( DBC ~ 40-50 mg/mL). You can remove Triton X 100 first . Then use TFF centrifugal / minimate / cassette / hollow fiber of appropriate molecular weight cut off (MWCO).
As your desire product MWCO and Urea < 1 kDa , You can use TFF devices to remove cyclodexin from the sample. even you can concentrate your protein.
Desire Molecular weight > 20 : 3kDa , 5kDa TFF devices
Desire Molecular weight > 50 : 5kDa , 10kDa TFF devices
Desire Molecular weight > 150 : 10kDa , 30kDa TFF devices
Detergent normally bind with TFF membranes. Please remove first with SDR hyperD sorbent followed by Diafiltration with TFF centrifugal device with buffer without Urea and triton X100. You can do ultrafiltration (concentration) of your product if you want.
Links;
1.SDR HyperD for detergent removal
2. TFF selection guide
3. Diafiltration application note.
Hope this works. For further assistance please write me on [email protected].
http://www.pall.com/main/laboratory/product.page?id=39374
http://www.pall.co.in/main/laboratory/literature-library-details.page?id=7046
http://www.pall.com/pdfs/Laboratory/02.0629_Buffer_Exchange_STR.pdf
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