I need to check protein expression via SDS, therefore I wanted to know the exact procedure of IPTG induction. whether I give induction in an overnight grown culture or should I go for secondary culture, and after how many hours of induction I need to take the sample for SDS. Please help if anyone knows
Hi there,
it depends on the temperature! At 37°C, a few hours of induction on actively growing cells is sufficient. What you could do in a first move is to follow induction by withdrawing aliquots for SDS/PAGE analysis (time 0, 1h, 2h, 3h...).
But if induction is at low temperature then induction could last overnight .
Dear Faiza Quddus
the induction protocol depends from your media AND culture volume.
If you are using LB in general you can growth an Overnight preculture (non inbduced), dilute in the morning it at least 1:100 in a fresh LB media carring antibiotics, growth the cells at 37°C up to OD(600nm) of about 0,6-0,8 and then induce with IPTG. THe lenght of the induction may change on the basis of induction temperature, eg. 3h at 37°C, 5h at 25°C or O/N at 17°C.
Lower temperatures may be usefull to improve protein solubility of stability.
you can find some information about in on the following link;:
https://www.blogger.com/video.g?token=AD6v5dxhZKT6Tvug0rLIZgcg8nyl5CwzQSFiX9pLglN-foSxrhnBjoEr7ReFTWj5bbeiloMCNhNoJn2HgHEgR2eC7SYwl5TPVwlCYNniPhSpeELHsevHk1dREBGIdHqskofd9HgX-2gr
best
Manuele
