5 replies to this topic

[DrAnt1]

Hi everyone,

I am running a time course experiment (8 time points in the 1st 24hrs and then one at 48 and one at 72hrs) dosing my cells with various things... I want to do nuclear protein extractions on these time points (I use the Active Motif kit) - as I have 8 time points (including 2 very anti-social points!) I do not wish to do individual extracts immediately following the time point (especially given each extract takes ~2hrs to process, not at all practical!) BUT I can't find any description of a method to store cell pellets to perform nuclear extractions all together at a later date.

If I were to spin down and freeze the cell pellet from each time point, is there an established method of defrosting them that will not damage the cells and allow efficient nuclear extraction?

If anyone has done nuclear extractions on a time course and can give me some Top Tips, it would be appreciated ... being still in the lab at 3am doing a single nuclear extraction is not an attractive option!!  

Thanks for your help!

[laurequillo]

I just pellet the cells and freeze them at -150. Then I start the protocol as usual

[DrAnt1]

laurequillo, on Feb 9 2010, 03:04 PM, said:

I just pellet the cells and freeze them at -150. Then I start the protocol as usual

Cool, thanks for replying!

I also received a reply from an ex-colleague who recommended the following (in case anyone else ever has this as a question!):

1. Terminate the cell culture at each desired time point by washing monolayer cultured cells with ice-cold PBS 2-3 times (can also trypsinise, add ice-cold media, pellet in cold centrifuge and wash with cold PBS 2/3 times using cold centrifuge)
2. Remove the last wash PBS thoroughly and snap-freeze the culture plate/pellet at -20C.

To perform extraction:
1. Take the plates out of the freezer and put them on ice (keeping them cold is very important).
2. Add appropriate amount of freshly made lysis buffer containing protease cocktails, then defrost and follow the procedure required for nuclear protein extraction.

[laurequillo]

DrAnt1, on Feb 11 2010, 06:42 AM, said:

laurequillo, on Feb 9 2010, 03:04 PM, said:

I just pellet the cells and freeze them at -150. Then I start the protocol as usual

Cool, thanks for replying!

I also received a reply from an ex-colleague who recommended the following (in case anyone else ever has this as a question!):

1. Terminate the cell culture at each desired time point by washing monolayer cultured cells with ice-cold PBS 2-3 times (can also trypsinise, add ice-cold media, pellet in cold centrifuge and wash with cold PBS 2/3 times using cold centrifuge)
2. Remove the last wash PBS thoroughly and snap-freeze the culture plate/pellet at -20C.

To perform extraction:
1. Take the plates out of the freezer and put them on ice (keeping them cold is very important).
2. Add appropriate amount of freshly made lysis buffer containing protease cocktails, then defrost and follow the procedure required for nuclear protein extraction.

Yeah that´s the way. (in my previous post I meant pellet the cells, remove the PBS/medium and freeze them, not to freeze them with the medium!!   )

[jokhf]

laurequillo, on 08 February 2010 - 09:04 PM, said:

I just pellet the cells and freeze them at -150. Then I start the protocol as usual

hi, im sorry, but what do you mean by pellet, are they completely dry in pellet form or are they in a pellet but also in buffer

[memari]

You can keep intact nuclear or  extract of nuclear in -80 without any problem.

I use this protocol without problem:

Panomics and Affymetrix Nuclear Extraction Protocol
http://babakmemari.f...-extraction.pdf

http://www.panomics....UCLEAR_1_V2.pdf