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after protein purification


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4 replies to this topic

#1 marzieh

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Posted 04 May 2015 - 05:34 AM

hello.

What process should be done after purification using IMAC method and before assessing the effect of the purified protein? are removal of imidazole and salt and protein concentration necessary? if yes, which methods are routinely used for these purposes?

thanks.



#2 phage434

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Posted 04 May 2015 - 07:19 AM

Many choices.  Easiest, perhaps, are ultrafiltration spin columns, which retain proteins and pass other components.  Slower, cheaper, and perhaps more effective is dialysis. Gel filtration can also be used. It depends a lot on what you want to do next, and how pure you need your protein.



#3 labtastic

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Posted 04 May 2015 - 09:06 AM

Desalting columns are fastest and easiest imo. Cheap and reusable.

 

Try GE Healthcare's PD-10 columns for samples up to 2.5ml. Or their Nap5 columns for up to 1ml samples. They take about 5 min to run once equilibrated with the proper buffer (which takes about 15-20min). >95% sample recovery, 99%+ removal of unwanted salts.

 

Or make your own column with G25 resin for larger samples.


Edited by labtastic, 04 May 2015 - 09:07 AM.


#4 marzieh

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Posted 07 May 2015 - 04:38 AM

Thanks for your helps.

i want to know generally that What process should be done after purification using IMAC method and before assessing the toxic effect of the purified protein on cancer cell lines?



#5 phage434

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Posted 07 May 2015 - 06:08 PM

You'll have to tell more about just where you need your protein.  Is this just added to the culture medium?  Injected in the cell? Expected to go to the nucleus? Protein won't get into cells without special care, so unless your protein is toxic extracellularly, adding it to culture media will likely not have much effect.






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