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Help With Lysing HEK293 & Fibroblast Cells


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#1 labmate1000

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Posted 02 August 2011 - 12:17 PM

Hi,

I am currently transfecting my cells (HEK293) with a GFP construct, and have been lysing them with RIPA buffer. The cell lysate is then read on a plate reader to detect fluorescence. My results aren't great, and I was wondering if anyone has experience in similar experiments? I don't think RIPA buffer is the most ideal lysis buffer, so any suggestions would be greatly appreciated.

I will also be transfecting human fibroblast cells soon to conduct the same experiment to measure GFP fluorescence. I have never transfected fibroblasts before, and I have been told they are much more difficult to transfect than HEK293s. Does anyone have advice on a good transfection reagent to use on fibroblasts?
In addition, I am not sure if RIPA buffer will be effective against fibroblasts, so any advice would also be greatly appreciated.

Sorry for all the questions and thanks in advance for all the help!

#2 bob1

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Posted 02 August 2011 - 05:29 PM

RIPA buffer is pretty good at lysis. You could try adding more detergents to help lyse the cells, but I don't think you will find them much more effective.

PEI and fugene seem to be quite effective for most cell types. YOu can use electroporation if you get really stuck, but this has the side effect of killing quite a lot of cells.




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