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the same construct, two stable cell clones with different protein size


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#1 Martha S

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Posted 26 September 2014 - 12:39 AM

 Hi,

I hope somebody can help me.

We transfected cells with a construct. After antibiotic selection we obtained many cell clones.

We made a real-time PCR and chose the best clones for further analysis.

Then we made a Western blot... What we see is surprising: two clones have a small protein band size (around 50 kDa), and all the rest bigger (around 80 KDa). The clones with the small size protein react to our experimental conditions in a differnet way as all others.

What happened? posttranslational modification? alternative splicing???

 



#2 Bio-Lad

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Posted 26 September 2014 - 06:10 AM

What construct are you transfecting?  What protein is being expressed?  What cells are you using?


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#3 Martha S

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Posted 30 September 2014 - 03:05 AM

Hey!

we are using pcDNA3 to overexpress a protein, which can have different splice variants. we inserted the full length sequence under pcDNA3.we use rat cell line.

we expect a protein band around 80 kDa.The WB is done under reducing conditions.

we obtained many stably transfected clones (confirmed by real-time RTPCR) and analyze the protein expression by Western blotting.

I do not understand that we've got some cell clones with 80 kDa and some with 50 kDa protein...

Why in the same basal cell line after transfection with the same plasmid we get two different (?) proteins?






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