I am looking to produce a phosphorylated protein using the TKX1 cells. sometimes I am getting the protein, but some times I cannot. I have few questions like does IPTG concentration effects the protein production?. For extracting the protein I use Freeze thaw technique. after freeze thaw if the cell lysate is too viscous I go for sonication and give 10-25 pulses at 1 sec interval. These days, I am not going for the sonication because cell lystae is not viscous. My post doc is saying that sonication is the one making the difference. I cant figure it out. Another thing is abt IPTG. In the manual it says induce the protein production with 0.1mM IPTG, usually I do with 0.6mM, my post doc is saying to use 1mM. Does the IPTG concentration effects the protein production? any type of help is appreciated.