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protein extraction - from cell line in T25 flask (Aug/05/2009 )

Hi,

I need to extract protein from my cell line in T25 culture flask and teh cells are 60-70% confluent. With this much confluency, how much RIPA shall I add. Also, cai I use the proteins for western immediately or would it be necessary to store in -80deg fro 24hrs? Any advice would be appreciated.

-SF_HK-

SF_HK on Aug 5 2009, 01:54 AM said:

Hi,

I need to extract protein from my cell line in T25 culture flask and teh cells are 60-70% confluent. With this much confluency, how much RIPA shall I add. Also, cai I use the proteins for western immediately or would it be necessary to store in -80deg fro 24hrs? Any advice would be appreciated.


Depending upon what protein concentration you want to work with, and confluency, the volume of lysis buffer I would use range from 100 ul to 500ul for a T25 flask. Of course, it would be best to use cell scraper for maximum yield.

You can store the lysate for western at -80 for an extended period of time, not only 24 hours. But don't forget to add protease inhibitor cocktail and PMSF. And if you are interested in phosphorylation states too, use phosphatase inhibitor too.

-cellcounter-

cellcounter on Aug 5 2009, 03:31 PM said:

SF_HK on Aug 5 2009, 01:54 AM said:

Hi,

I need to extract protein from my cell line in T25 culture flask and teh cells are 60-70% confluent. With this much confluency, how much RIPA shall I add. Also, cai I use the proteins for western immediately or would it be necessary to store in -80deg fro 24hrs? Any advice would be appreciated.


Depending upon what protein concentration you want to work with, and confluency, the volume of lysis buffer I would use range from 100 ul to 500ul for a T25 flask. Of course, it would be best to use cell scraper for maximum yield.

You can store the lysate for western at -80 for an extended period of time, not only 24 hours. But don't forget to add protease inhibitor cocktail and PMSF. And if you are interested in phosphorylation states too, use phosphatase inhibitor too.


On a related note, can you store a cell lysate at -80 before performing a co-immunoprecipitation or will freezing damage the proteins so that they will no longer interact?

Thanks,
P

-Penguin-

Penguin on Aug 6 2009, 09:32 AM said:

cellcounter on Aug 5 2009, 03:31 PM said:

SF_HK on Aug 5 2009, 01:54 AM said:

Hi,

I need to extract protein from my cell line in T25 culture flask and teh cells are 60-70% confluent. With this much confluency, how much RIPA shall I add. Also, cai I use the proteins for western immediately or would it be necessary to store in -80deg fro 24hrs? Any advice would be appreciated.


Depending upon what protein concentration you want to work with, and confluency, the volume of lysis buffer I would use range from 100 ul to 500ul for a T25 flask. Of course, it would be best to use cell scraper for maximum yield.

You can store the lysate for western at -80 for an extended period of time, not only 24 hours. But don't forget to add protease inhibitor cocktail and PMSF. And if you are interested in phosphorylation states too, use phosphatase inhibitor too.


On a related note, can you store a cell lysate at -80 before performing a co-immunoprecipitation or will freezing damage the proteins so that they will no longer interact?

Thanks,
P



I found that my IPs worked better when the lysates were used fresh rather than stored at -80C, but this may have been a coincidence. I never directly compared side by side IPs from fresh vs. frozen lysates.

-Dr Teeth-

Penguin on Aug 6 2009, 05:32 AM said:

cellcounter on Aug 5 2009, 03:31 PM said:

SF_HK on Aug 5 2009, 01:54 AM said:

Hi,

I need to extract protein from my cell line in T25 culture flask and teh cells are 60-70% confluent. With this much confluency, how much RIPA shall I add. Also, cai I use the proteins for western immediately or would it be necessary to store in -80deg fro 24hrs? Any advice would be appreciated.


Depending upon what protein concentration you want to work with, and confluency, the volume of lysis buffer I would use range from 100 ul to 500ul for a T25 flask. Of course, it would be best to use cell scraper for maximum yield.

You can store the lysate for western at -80 for an extended period of time, not only 24 hours. But don't forget to add protease inhibitor cocktail and PMSF. And if you are interested in phosphorylation states too, use phosphatase inhibitor too.


On a related note, can you store a cell lysate at -80 before performing a co-immunoprecipitation or will freezing damage the proteins so that they will no longer interact?

Thanks,
P

Co IPs fare worse or do not work at all when it is done using lysates stored at -80. Always try to use the freshly made lysate for this purpose.

The reasons (I think) are that..

(1) during the freeze-thaw procedure, the complexes that may have formed may break-up and may not re-form during the overnight incubation.

(2) there is also this thing that certain proteins do not come back into solution completely after thawing your lysates, and if those proteins happen to be your protein or binding proteins thereof, you would not get good results.

If you must use such frozen lysates, I suggest thawing them on ice for a long time, rather than expediting the process by leaving them at RT.

-cellcounter-

cellcounter on Aug 6 2009, 04:35 PM said:

Co IPs fare worse or do not work at all when it is done using lysates stored at -80. Always try to use the freshly made lysate for this purpose.

The reasons (I think) are that..

(1) during the freeze-thaw procedure, the complexes that may have formed may break-up and may not re-form during the overnight incubation.

(2) there is also this thing that certain proteins do not come back into solution completely after thawing your lysates, and if those proteins happen to be your protein or binding proteins thereof, you would not get good results.

If you must use such frozen lysates, I suggest thawing them on ice for a long time, rather than expediting the process by leaving them at RT.


Yeah, that's what I suspected but I thought I'd ask in case I could make life a bit easier.... :)
P

-Penguin-