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Western blot of apoptotic cells


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#1 sixlayers

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Posted 06 April 2011 - 08:03 AM

Hi all,
The procedure I normally use for western blot is as follows: I have the cells in 6-well plates (each well having equal numbers of cells) incubated with the compound of interest for a certain time. Then I wash the cells 2x with PBS buffer before adding lysis buffer and scraping the cells off.

The problem now is that I am testing a compound that is inducing apoptosis in cancer cell lines (MCF-7) in a concentration dependent way. The apoptotic cells (so especially the wells with higher concentration of the compound) will float away and if I wash them I loose them. I only analyse the attached cells not the floating cells. Therefore, I get a weaker signal for the wells with higher concentration of compound.

What is the proper way to retrieve all cells from the 6-well plates? Any advice is much appreciated!

#2 bob1

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Posted 06 April 2011 - 07:08 PM

You can collect the lifted off cells in a tube and then lift cells with trypsin (or other agent), spin down, wash in PBS, then spin again etc.

#3 sixlayers

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Posted 07 April 2011 - 01:01 AM

Hmm... after you said it, it seems to be kind of obvious :) Thank you very much for your help!!




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