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dachshund's Content

There have been 4 items by dachshund (Search limited from 05-April 19)


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#84160 RT-PCR contamination issue

Posted by dachshund on 19 August 2010 - 05:44 PM in PCR, RT-PCR and Real-Time PCR

I am experiencing the same problem~ Have you got any luck so far?



#42629 how to deal with protein precipitates during dialysis

Posted by dachshund on 04 November 2009 - 05:18 PM in Protein and Proteomics

Hi all:

I am now trying to purify my His-tagged protein. Some of the protein precipitated during dialysis, my next step is to cleave the protein using EKmax protease. It seemed like that the protease did not digest the precipitated proteins. What should I do with those precipitates? Any suggestion is appreciated!



#37394 help~ how concentrate protein solution in large-scale

Posted by dachshund on 23 September 2009 - 08:01 PM in Protein and Proteomics

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~



#36501 help~ how concentrate protein solution in large-scale

Posted by dachshund on 15 September 2009 - 01:28 PM in Protein and Proteomics

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~




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