I did a trial run of a his-purification and found that my protein would be a little dilute for my purposes. Any thoughts on the best way to concentrate a denatured protein?
This is my buffer and protein...
10mM Phosphate pH 7.4, 500mM NaCl, 500mM Imidiazole, 8M Urea
~35kDa
I'm leaning to ultra-filtration since it will likely leave most of my protein in solution. I'd rather not let it fall out if I can help it. I will be going to dialysis next, so maybe I could perform a two in one!
Thoughts?
A 35 kDa protein should not pass through a 10 kDa filter. What went through may be small proteins that are contaminants in your protein sample, or your protein may have suffered proteolysis, or the filter may have been broken. Did you run some of the flow-through on a gel to see if it is your protein?
Ultrafiltration could induce protein precipitation. I would first remove urea slowly using dialysis, afterwards I would concentrate using ultrafiltration.
I regularly use Vivaspin 20 columns to concentrate my protein (Amyloid-beta peptide 4.5 kDa, very soluble in 8M Urea). It takes a few hours for the urea to run through the column, but it works.
But, you should have a look if your protein precipitates in those conditions.
Just a follow up.
I tried concentrating my trial sample with the Amicon Ultra 10 NMWL and it concentrated my protein without precipitation. However I did observe a lot of protein in the flow-through. (~30% retention) I think I will look into HIC, Anion Exchange, and Amicon Ultra 5 NMWL as a means of concentrating my protein. Any other ideas would be appreciated while I am in the planning stage.
Thank you.
A 35 kDa protein should not pass through a 10 kDa filter. What went through may be small proteins that are contaminants in your protein sample, or your protein may have suffered proteolysis, or the filter may have been broken. Did you run some of the flow-through on a gel to see if it is your protein?
Hello,
I would suggest you to go for dialysis first, and then use PEG for concentration of protein. Lyophilisation can also be done, and then you can dissolve your protein ....
Good Luck...
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques