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Transfection help

Transfect with HEK 293

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#1 Vanessa Walters

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Posted 26 December 2016 - 06:09 PM

Hello everyone,

 

I am currently having problems with my transfections and request help. I use lipofectamine 3000 to transfect Hek 293 cells in 6 wells plates. For the last transfection, I transfected at 60% confluency. Also, I used 2500 ng of DNA per well, 5µl of P3000 per DNA, 5µl of Lipofectamine 3000, and DMEM with P/s + L-glu instead of OMEM. However, after 48 hours of transfections majority of the cells were lifted off the plate. However, even though the cells detached from the plate and little pellets remained after they were centrifuged, the lysate showed protein expression but not the medium (shown from western western analysis). But the problem is  that I need protein to also express in the medium. Even when I incubated the transfected cells for 72 hours and used OMEM, there still isn't protein expression shown in the medium.

 

Any advice or recommendations will be greatly appreciated. 



#2 bob1

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Posted 27 December 2016 - 06:55 AM

You can take one thing from the above - you have expression of your protein. However, you don't see it being secreted into the medium as you would like. So, the things to look at are:

 

Does the protein reach the cellular membrane of the cells? You can test this by immunocytochemistry.

How strong is your expression? Can you run and stain (coomassie) a gel and see a difference in the bands that might be your overexpressed protein.

Does your plasmid have the correct components to make the cell translocate the protein to the surface and secrete it? You will need a signal peptide to get it to the ER and then translocated. If you have one of these, have you tried others, these can make a big difference in expression?

Have you tried other cell lines? 293 are easy to transfect but notorious for lifting off the plate, you might find that CHO or another cell line works better for you.

You can try titrating the amount of DNA - more is not necessarily better for transfections. I have often found that 0.5-1 ug per well of a 6 well plate was better or just as good as 2 ug or more.

Is the overexpression causing the cells to be unhappy/die? 






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