The lysis buffer will be diluted by the LB. If there is a component in the lysis buffer for which the concentration is of high importance, then the total volume of the sample including the LB should be taken into account when deciding how much lysis buffer to add.

Dear Jonh

if your protein is expressed in the citoplasm and you would like to perform total cell lysis you can store e.coli pellets at -20 for years so dont worry about some days. If your protein of expressed in periplasm and you would like to selectivelly extract it for example with osmotic shock, froze the cells is not reccomended because yiu can induce some Total cell lysis and contaminate the periplamsic extract with some citoplamic proteins.

however the next time i suggest to you to discard all the lb medium after centrifugation and store only the wet pellet because of course now the lb will be mixed with your extraction/lysis buffer and in some cases ( depends from lysys and purification approach that you wilk use) it can disturb the performances of your folllwing steps

(eg lb contain salt and you have to dilute it a lot to decrease the Salt concentration if you would like to perform a ionic exchange and increase a lot the resuspension volume may reduce the efficience of your lysis approach and make the sample load in the coloumn a long process.

good luck

ciao

Manuele

It's quite fair to keep the cells frozen. If you don't sonicate or lyse prior to storage, its safe to use cells for extracting enzyme or protein of interest. You may resuspend the cells after storage period in PBS, centrifuge and add lysis buffer or sonicate.

I don't think you should be paranoid since the pellet is already now frozen. However, prior to addition of lysis buffer for sonication process, a resuspension and centrifugation at mild speed is advised to remove the excess LB.

@adam b Shapiro thank you for your answer and all following answers.

Our protocol does not make clear the thawing process. What is the common/best method to ensure cell integrity during this step? Premature lysis will mean protein in solution during the sonication step leading to denaturation of the protein. I have heard 37 degrees in a water bath

Dear Jonh

if you store the wet cell pellet after centrifugation with out the media, at the moment of the purification you can just resuspend the cells in the lysis buffer by vortex at room temperature, move it in ice and sonicate the resuspended sample.

the problem of your sample not sokid But resuspended in lb is that at -80C you can have some cell lysis and if now you (as adeyemi suggest) will perform a centrifugation step at mild speed to remove the lb, you may lost a fraction of your protein extracted in lb from the cells the were lysate by the frozing step, so as Adam suggest at this point the best approach is de-froze your samples (at 4C or rt), dilute directly it with lysis/binding buffer, and perform the sonication in ice.

the volume and composition of lysis buffer that you need to add depend from the purification tecnique that you would like to use.

ciao

Manuele Martinelli

My preferred method is to resuspend the still-frozen pellet in the lysis buffer while it is thawing. Once it is just barely thawed, I homogenize the suspension with a loose-fitting glass Dounce homogenizer. (Probably not necessary if you are going to sonicate it. I use a French Press rather than sonication.) I don't think the proteins suffer denaturation when they are sonicated, even if the cells are already broken, as long as the sonication is done properly, i.e. no foaming and the sample is not allowed to warm up very much. If you have the suspension in a steel beaker set in ice/water slurry during sonication, heat is conducted out of the sample better than if you use a glass or plastic container.