Dear all,
I am going to analyze the proteins of whole cell. The cells are cultured on surface of T-flask. usually, I use Trypsin to harvest cells. However, it is said ENTA is better since it did not destroy the surface proteins.
Anyone can tell me how does EDTA work? I want to know the mechanism.
Thank you!!
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#2
badcell
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Posted 07 January 2005 - 07:41 AM
EDTA acts by chelating calcium and magnesium ions from the intercellular bridges and desmosomes. Cells then dissociate from each other and from the support media. You can use 1-2% EDTA diluted in HBBS or PBS, the incubation time will depend on your cell line. EDTA is not highly efficient by itself, but potentiates the action of trypsin.
If you want to obtain intact cells you may also try scraping them. I usually trypsinize the clls for subculturing but scrape them when I'm gonna extract protein. Cheers!
If you want to obtain intact cells you may also try scraping them. I usually trypsinize the clls for subculturing but scrape them when I'm gonna extract protein. Cheers!
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#3
quanzeng
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Posted 07 January 2005 - 01:30 PM
Thank you for your reply!
#4
themoon
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Posted 07 December 2009 - 02:56 PM
badcell, on Jan 7 2005, 07:41 AM, said:
EDTA acts by chelating calcium and magnesium ions from the intercellular bridges and desmosomes. Cells then dissociate from each other and from the support media. You can use 1-2% EDTA diluted in HBBS or PBS, the incubation time will depend on your cell line. EDTA is not highly efficient by itself, but potentiates the action of trypsin.
If you want to obtain intact cells you may also try scraping them. I usually trypsinize the clls for subculturing but scrape them when I'm gonna extract protein. Cheers!
If you want to obtain intact cells you may also try scraping them. I usually trypsinize the clls for subculturing but scrape them when I'm gonna extract protein. Cheers!
I have really sensitive primary cells. I've tried 0.04% EDTA with no luck. They just don't want to come off. And I don't want to use trypsin since it may compromise the cell surface proteins.. Would 1%EDTA be too much for them? How well do they come off with 1%?
Thanks!
#5
lab rat
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Posted 07 December 2009 - 03:57 PM
2 mM EDTA in PBS, pH 7.4, is sufficient to remove adherent cells, but this depends on the cell type. Some of my cell lines require incubation at 37C for a few minutes before liftoff, others require a sharp smack to come off the flask.
Trypsin will damage your surface proteins, but you may still need it to remove cells from the flask.
Trypsin will damage your surface proteins, but you may still need it to remove cells from the flask.
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#6
themoon
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Posted 09 December 2009 - 04:50 PM
lab rat, on Dec 7 2009, 03:57 PM, said:
2 mM EDTA in PBS, pH 7.4, is sufficient to remove adherent cells, but this depends on the cell type. Some of my cell lines require incubation at 37C for a few minutes before liftoff, others require a sharp smack to come off the flask.
Trypsin will damage your surface proteins, but you may still need it to remove cells from the flask.
Trypsin will damage your surface proteins, but you may still need it to remove cells from the flask.
thanks!!
