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  2. → Viewing Profile: Posts: jameela

jameela

Member Since 08 Oct 2012
Offline Last Active Oct 25 2012 03:34 AM
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Posts I've Made

In Topic: cloning a gene in to a plasmid

25 October 2012 - 03:34 AM

the protein is narmally expressed in the hek cells but not the myc tagged ones, i will try the ELISA and see.
thank you very much

In Topic: cloning a gene in to a plasmid

23 October 2012 - 11:56 AM

Hello,
I have an other question regarding transfection, I transfected HEK293 cells with plasmid that carries my gene, and I selected cells that are stabelly expressing my gene by antibiotic selection, the protein that I am interested in studying should be execreted in the media of the cells , so in order to be sure that I have my protein I preformed a westren blot using a MYC antibody as my protein was myc tagged and i used the media of my protein directly , the problem is that every time i do a westrn blot i get the same pattern of bands even in control sample that was not transfected,one of the bands is in the right size place but i dont trust this result,i tried using my protein antibody i get the same result? any idea about what is happning?

In Topic: cloning a gene in to a plasmid

10 October 2012 - 01:09 AM

Thank you very much bob1..and yes my plasmid that i use is 5.5KB with out the insert but the commercial one is 3.5 together with the insert!
and i use DPN1 after SDM it works perfectly with the commercial plasmid , where usually a sharp band of the same size of plasmid sti exsists after DPNI treatment, but with my insert i get many faint bands that simply disapear completly after DPN1 treatment...!
ill try your suggestion it sounds very logical thank you

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