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help~ how concentrate protein solution in large-scale


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#1 dachshund

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Posted 15 September 2009 - 01:28 PM

Hi, everyone~
I've just finished my protein expression in 2L prep, because the proteins were presented in inclusion bodies, I added denaturing buffer which contained 6M Guanidine hydrachloride to solubilize the protein. The problem was I added way too much denaturing buffer, and this made the following purification step became very complicated. I tried to concentrate the solution using diafiltration method, but it did not work, and my supervisor said I might damage the membrane already... :(

Can anyone give me any suggestions? All of them are appreciated~! Thank you in advance~

#2 BIOKMST

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Posted 17 September 2009 - 11:44 AM

Hi, everyone~
I've just finished my protein expression in 2L prep, because the proteins were presented in inclusion bodies, I added denaturing buffer which contained 6M Guanidine hydrachloride to solubilize the protein. The problem was I added way too much denaturing buffer, and this made the following purification step became very complicated. I tried to concentrate the solution using diafiltration method, but it did not work, and my supervisor said I might damage the membrane already... :)

Can anyone give me any suggestions? All of them are appreciated~! Thank you in advance~



You are in 2 ltrs? Wow. Can you bind your protein to any chromatography column? If you have a lot of ammonium sulfate, you can pellet it and then dialyze the salts out.

#3 dachshund

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Posted 23 September 2009 - 08:01 PM

Hi, everyone~
I've just finished my protein expression in 2L prep, because the proteins were presented in inclusion bodies, I added denaturing buffer which contained 6M Guanidine hydrachloride to solubilize the protein. The problem was I added way too much denaturing buffer, and this made the following purification step became very complicated. I tried to concentrate the solution using diafiltration method, but it did not work, and my supervisor said I might damage the membrane already... :unsure:

Can anyone give me any suggestions? All of them are appreciated~! Thank you in advance~



You are in 2 ltrs? Wow. Can you bind your protein to any chromatography column? If you have a lot of ammonium sulfate, you can pellet it and then dialyze the salts out.


Yes, I did 2L prep to express my isotope labeled protein for NMR analysis. My protein is His-tagged, so yes, I can purify them using Ni coated IMAC column. However, due to large amount of very diluted protein solution, I ran the whole purification for 2 weeks.... My next step is to refold the protein and desalt the solution using TFF diafiltration system~
Thanks for the post~




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