Hey Dilip, There are many reasons for this observation. Rafael is right, please do share the image of the gel. Also what are the exact sizes of the two proteins that you co-expressed? I have observed the heterodimeric and pentameric proteins on SDS-PAGE albeit the presence of DTT in the sample buffer, and despite boiling of the samples. Could this be that 60 KD is actually the hetero-dimer? One can perform western blotting, followed by the use of anti-polyhistidine and anti-Streptavidin antibodies on separate blots to confirm the suspected bands. Likewise SEC followed by WB may confirm the identity of the eluted proteins in different fractions after size exclusion chromatography. You may also cleave the tags, and then see the magic!
Tom has correctly pointed out that induction at lower temperature is best achieved upon incubation of culture so that the temperature is dropped before the induction. Bon Voyage! -Z Zaigham M Khan, PhD Associate Scientist Icahn School of Medicine at Mount Sinai Department of Oncological Sciences 1470 Madison Avenue New York United States On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar <[email protected]> wrote: > 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 > ######################################################################## 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/
