You may want to check out the Studier paper on autoinduction from 2005. It has various bits of advice about SeMet labelling, like getting the amino acid mixture right and vitamin/metal stocks. I follow this and do fine with BL21 DE3 cells.
Jon On Thu, Jul 03, 2014 at 10:00 PM, Maher Alayyoubi <maher.alayyo...@gmail.com<mailto:maher.alayyo...@gmail.com>> wrote: Hi Everyone, Can someone please help me figure out what's wrong with my SelMet incorporation protocol. My protein of interest is 500 amino acids with 20 of these being Methionines. However mass spec data show that only two of these 20 Methionine sites are partially labelled with SelMet. The following is the labeling protocol that I used: - Inoculate 5ml of LB media with a T7 Express LysY/Iq cells transformed with my plasmid - Let shake at 37C for 10 hours - Spin down - Wash with 5ml minimal media - Spin down again - Suspend in 5ml minimal media and add to 100ml minimal media overnight at 37C - Next day transfer 20ml from the overnight 100ml minimal media culture to 0.5L of minimal media - Let grow until OD of 0.6 at 37C - Then add: 100mg Lysine, 100mg phenylalanine, 100mg threonine, 50mg isoleucine, 50mg leucine, 50mg valine, and 60mg Selenomethionine - Let shake for 30min - Then add 1mM IPTG and let shake at 18C overnight. Thank you very much, Maher -
