2 replies to this topic

[azrael201]

Is there a proper/logical way to freeze my cell pellets to save them for immunoblotting later?

Currently my understanding is to trypsinize and wash with PBS and then spin down as packed as possible as to aspirate all the PBS from the cells. I think in a large Sorvall rotor I do 10 minutes at approximately 1500xg that way I maximize PBS aspiration while minimizing cell loss. Then freeze in -80?

However, I noticed when I thaw them on ice for about 5-10 minutes and attempted to resuspend in PBS to transfer to microcentrifuge tubes I noticed there was viscous blobs in some of the tubes that most likely meant premature cell lysis. I could not resuspend the blobs in lysis buffer and had to sonicate.

How can I minimize that premature lysis is that from inadequate aspiration of PBS prior to freezing?

Also my postdoc said I should have added the lysis buffer directly to the 15ml tubes instead of transfering them first to add the lysis buffer. Will that really make a large difference in my protein concentration?



Thanks!

[protolder]

azrael201, on 22 March 2011 - 08:28 AM, said:

Is there a proper/logical way to freeze my cell pellets to save them for immunoblotting later?

Currently my understanding is to trypsinize and wash with PBS and then spin down as packed as possible as to aspirate all the PBS from the cells. I think in a large Sorvall rotor I do 10 minutes at approximately 1500xg that way I maximize PBS aspiration while minimizing cell loss. Then freeze in -80?

However, I noticed when I thaw them on ice for about 5-10 minutes and attempted to resuspend in PBS to transfer to microcentrifuge tubes I noticed there was viscous blobs in some of the tubes that most likely meant premature cell lysis. I could not resuspend the blobs in lysis buffer and had to sonicate.

How can I minimize that premature lysis is that from inadequate aspiration of PBS prior to freezing?

Also my postdoc said I should have added the lysis buffer directly to the 15ml tubes instead of transfering them first to add the lysis buffer. Will that really make a large difference in my protein concentration?



Thanks!

Hola, you could store your cells as cellular stocks at -80 with dmso and follow instructions for freezeng and tawing. or after wash, add the lysis buffer and keep at -80 untill use. viscosity ids due to DNA so or you sonicate and load hor directly from the heater block. Buena suerte

[bob1]

Personally I would freeze them in lysis buffer, unless you are wanting to do sub-cellular fractionation, in which case don't freeze them!