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Freezing cells before lysis? - (Mar/05/2008 )

Hi,
I am new to cell culture work and am working with HeLa and HEK293 cells and would like to harvest the cells (after several hours of nutrient limitation) and run an SDS gel the next day.

I can't lyse them and freeze the lysate as the protein of interest doesn't do well with freeze/thaw, and I should run the gel immediately after adding sample buffer/boiling.

Can I simply freeze the cells in N2 and resuspend in lysis buffer the next day? Or is there a freezing media that I should use and spin them out of the next day?

Thanks for your help.

-ailedroc-

QUOTE (ailedroc @ Mar 5 2008, 05:19 PM)
Hi,
I am new to cell culture work and am working with HeLa and HEK293 cells and would like to harvest the cells (after several hours of nutrient limitation) and run an SDS gel the next day.

I can't lyse them and freeze the lysate as the protein of interest doesn't do well with freeze/thaw, and I should run the gel immediately after adding sample buffer/boiling.

Can I simply freeze the cells in N2 and resuspend in lysis buffer the next day? Or is there a freezing media that I should use and spin them out of the next day?

Thanks for your help.


If you are only going to run the samples on SDS-PAGE then there should be no problem in lysing the cells and freezing, since the protein will be denatured anyway by the SDS. You could either:

- lyse the cells with lysis buffer, do protein determination, and then add SDS-sample buffer to the samples, boil (95degree C, 5 min) and freeze until further analysis

- lyse the cells directly with SDS-sample buffer, boil and freeze until further analysis

Hope this was of any help:)

-boxfish-

QUOTE (ailedroc @ Mar 5 2008, 09:19 AM)
Hi,
I am new to cell culture work and am working with HeLa and HEK293 cells and would like to harvest the cells (after several hours of nutrient limitation) and run an SDS gel the next day.

I can't lyse them and freeze the lysate as the protein of interest doesn't do well with freeze/thaw, and I should run the gel immediately after adding sample buffer/boiling.

Can I simply freeze the cells in N2 and resuspend in lysis buffer the next day? Or is there a freezing media that I should use and spin them out of the next day?

Thanks for your help.


do not freeze eukaryotic cells in N2 or without freezing medium as it will kill cells; thawing of killed cells will damage proteins as the lysosomes are disintegated;

freeze medium are special for each type of cell; use a freezing box with isopropanol for -80°C freezer...

-The Bearer-

Thanks for the advice!

-ailedroc-

QUOTE (boxfish @ Mar 6 2008, 09:12 PM)
QUOTE (ailedroc @ Mar 5 2008, 05:19 PM)
Hi,
I am new to cell culture work and am working with HeLa and HEK293 cells and would like to harvest the cells (after several hours of nutrient limitation) and run an SDS gel the next day.

I can't lyse them and freeze the lysate as the protein of interest doesn't do well with freeze/thaw, and I should run the gel immediately after adding sample buffer/boiling.

Can I simply freeze the cells in N2 and resuspend in lysis buffer the next day? Or is there a freezing media that I should use and spin them out of the next day?

Thanks for your help.


If you are only going to run the samples on SDS-PAGE then there should be no problem in lysing the cells and freezing, since the protein will be denatured anyway by the SDS. You could either:

- lyse the cells with lysis buffer, do protein determination, and then add SDS-sample buffer to the samples, boil (95degree C, 5 min) and freeze until further analysis

- lyse the cells directly with SDS-sample buffer, boil and freeze until further analysis

Hope this was of any help:)

i think you can freeze the cell at -80 degree for couple of weeks. then do the lysis.

-party-