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Control protein for eukaryotic expression


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5 replies to this topic

#1 Rafmac

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Posted 04 October 2013 - 01:02 AM

Hi Guys,

 

I am new to the forum, and so do I in recombinant protein expression in eukaryotic cells :-)

I am trying to set-up a recombinant protein expression in my lab, with HEK293 cells and CHO cells (expression vector: pcDNA3). For the moment I will try transient expression.

I was wondering if someone could advice to me a good positive control (I mean a protein) to validate my culture and transfection procedure. I mean, can someone tell which protein will be expressed for sure with pcDNA3/HEK293 or pcDNA3/CHO ?

Thanks a lot for your help.

 

PS: sorry for my poor english.

 



#2 GNANA

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Posted 04 October 2013 - 06:07 AM

If you wish to check the transfection efficiency you can use GFP driven by the same promoter as pCDNA3.


I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 Rafmac

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Posted 07 October 2013 - 04:15 AM

Hi GNANA,

 

Thanks for your reply. The aim is not (only) to check tranfection efficiency, but also to check protein production/purification yield (after one-step IMAC).

Is the GFP expressed at good level ?


Edited by Rafmac, 07 October 2013 - 04:15 AM.


#4 IBMSterry

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Posted 09 October 2013 - 10:28 PM

Hi,

 i think you want to choose a protein to opyimize the purification system. so why not to choose an easily-done protein, such as GST. 

 best wishes.



#5 Rafmac

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Posted 11 October 2013 - 12:31 AM

Hi IBMSterry,

 

Thanks for your reply. Using a protein easy to purify is a good idea. Do you think that GST from pGEX vectors (S.japonicum) would be a good choice ?

Having that said, has anyone ever expressed a human recombinant in HEK293 with good expression rate ? To be honest, I would prefer this kind of control for my expression system.



#6 jerryshelly1

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Posted 11 October 2013 - 08:43 AM

HEK's are marvelous for expression studies. Whenever I have a problematic expression vector, I stick it into HEK's and see marvelous expression. HEK cells are a good choice.






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