I have heard of up to 15% ethanol being helpful, but have not tried that myself.
Addition of a cleavable MBP tag to the protein is an option. I have been working with some few difficult cases with respect to protein solubility on lysis. Diluting the lysis conditions, a little to a lot, seems to be the most promising strategy. Does anone else have experience with this approach? Also, does anyone have a preferred method for testing for whether the protein is in solution or already insoluble in the cell before lysis. I seem to be seeing that small scale standard lysis using freeze-thaw, followed by centrifugation, followed by addition of SDS-loading dye separately to the pellet and supernatant has falsely suggested insoluble protein, when actaully the lysis method was responsible. (I think...). sincerely, Anthony Duff ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of A K [alek6...@gmail.com] Sent: Wednesday, 27 February 2013 9:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Additives to bacterial cultures to improve solubility Mark, you can try low percentage of ethanol as well, as it increases the expression of some chaperones (can't find the paper right now!). Alex On Tue, Feb 26, 2013 at 2:08 AM, Mark J van Raaij <mjvanra...@cnb.csic.es<mailto:mjvanra...@cnb.csic.es>> wrote: Dear All, We are considering trying additives to bacterial cultures to see if they improve solubility of some proteins we express: L-arginine, glycyl-glycine, sorbitol and betaine. I would be glad to receive recommendations and experiences, also if they haven't worked. Side-question: should we get cell culture-certified compounds, or would "normal", much more economical ones suffice? (i.e. are they likely to contain side-products that would affect bacterial growth?). Greetings, Mark Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616<tel:%28%2B34%29%2091%20585%204616> http://www.cnb.csic.es/~mjvanraaij<http://www.cnb.csic.es/%7Emjvanraaij> -- David J. Leibly Graduate Student, Yeates Lab Dept. Chem & Biochem Univ. of California, Los Angeles Boyer Hall 269B (310) 825-8901
