2 replies to this topic

[anonymous]

For extracting protein from TBY2 cells, I use a typical protein homogenizing buffer such as 50 mM Tris or HEPES pH 7.5, 28mM B-Mercaptoethanol, 2mMEDTA, and 2mM PMSF.  You can add additional protease inhibitors such as antipain,6-aminocaproic acid, benzamidine, etc. if your protein is being degraded.  

For extraction of proteins I freeze tissues on liquid Nitrogen and grind them in a mortar with a pestle.  I then add about 10 mls of ice cold homogenizing buffer per 1 gram of tissue.  I have found that grinding plant tissues with a mortar and pestle works better than nitrogen cavitation or a dounce homogenizer due to the difficulty in breaking up the cell wall.  

Depending on what protein you are looking at, you may want to try a couple of different time points.  For example you may want to try tissue samples from cultures 3-4 days after transfer if your protein is growth associated or 7 days after transfer if your protein expression is associated with a quiescent state.  If you want a more detailed protocol, send E-mail to me.  By the way, do you know how I can attain Tobacco BY-2 cells.  We have not used the cell line for quite some time now and I would like to do a few more experiments. Unfortunately, our cell line as ceased to exist.

Ken MaasDept. of Biological SciencesNorthern Illinois UniversityDeKalb, Illinois USA 60115kjmaas@niu.edu

[anonymous]

Hi, friends:Can anybody tell me how to extract crude protein and DNA from BY-2 cells? Thank you very much

[anonymous]

For quick-and-dirty preps we use a product called DNAzol ES <a href> [url="http://www.mrcgene.com/dnaes.htm"][url]http://www.mrcgene.com/dnaes.htm[/url][/url] </a href>, grinding the tissuein liquid N, and letting the DNA ethanol precipitate overnight.  This is pretty fastand easy, but there is always some lingering carbohydrate and RNA, soit is not clean enough for cloning.