Greetings, everyone I am trying to co-express two mammalian proteins (less than 50 kDa MW) in Rosetta cells but getting a contaminating band around 60 kDa. One of the construct is in pET28a with His tag while the other is in pET21c with Strep tag and I am adding all the three selection markers during growth of pre-culture and during induction. Initially, cells were grown at 37 °C till OD reaches to 0.6 then induced with 0.5mM IPTG and incubated at 16°C ON. When I do purification using Streptactin resin; I can see proteins of my interest bound to the resin along with contaminating protein at 60 kDa. I have tried performing size exclusion as a follow up step but they are co-eluting in void volume. I have also tried to wash with MgCL2-ATP solution but it co-elution with contaminant. I am looking for valuable suggestions to avoid the contamination during or after expression.
Thanks in advance. Regards Dilip ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
