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
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