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Rate of protein production with pcDNA 3.3 transfected into HEK293


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#1 Micro

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Posted 11 November 2013 - 02:33 PM

We have outsourced the production of a transfected cell line that will produce a protein of interest. My supervisor wants to know the protein production pattern of our target protein after transfection (i.e. log, linear, exponential). Since we don't have the ability to determine this ourselves, I was wondering if anyone knows a source or general rules of thumb for this type of information?

 

The details for a transfected cell line are as follows:

cell line = HEK293

plasmid = pcDNA 3.3

promoter = using plamsid's promoter 

protein size ~55kDa

protein stability= transient

 

Let me know if I need to provide more information. Any help would be greatly appreciated.unsure.png

 

M

 



#2 bob1

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Posted 11 November 2013 - 04:27 PM

Why don't you have the ability to do this? - all it takes is the ability to detect it on western blot  and some means of normalizing the blot to protein or cells loaded and a time course experiment.

 

In general though, most biological systems follow a sort of exponential curve where you have a lag phase before the plasmid is activated, and then slow production that increases as more and more plasmid copies come "on-line".  This will tend to level out after a while and give you some sort of steady state.



#3 Micro

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Posted 12 November 2013 - 01:45 PM

Thanks for your response smile.png



#4 Osibisa

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Posted 14 January 2014 - 08:15 PM

What sort of protein is it? I.e. intracellular and especially membrane proteins will be under tighter control than secreted proteins. You could consider using HEK293-T cells which will replicate the vector episomally due to the SV40 origin of replication.






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